Optimized in vivo delivery system with endosomolytic agents for nucleic acid conjugates

ABSTRACT

The present invention relates to an optimized in vivo delivery system with endosomolytic agents for nucleic acid of therapeutic interest conjugated to molecules facilitating endocytosis, in particular for use in the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/232,844, filed Aug. 10, 2016, now U.S. Pat. No. 9,687,557, which is acontinuation of U.S. application Ser. No. 14/923,482, filed Oct. 27,2015, now U.S. Pat. No. 9,428,538, which is a continuation of U.S.application Ser. No. 13/703,965, filed Feb. 5, 2013, now U.S. Pat. No.9,205,099, which is the U.S. national stage application of InternationalPatent Application No. PCT/EP2011/060280, filed Jun. 21, 2011.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Oct. 22, 2014 and is 19 KB. The entire content ofthe sequence listing is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to the field of medicine, in particularoncology.

BACKGROUND OF THE INVENTION

Cancer treatment mainly consists of surgery wherever possible, cytotoxicagents such as chemotherapy, and radiotherapy. Molecular therapies forcancer treatment have emerged in the last decade, such as monoclonalantibodies targeting the cell membrane receptors, and inhibitors oftyrosine kinase receptors and other kinases which target signaltransduction involved in cell proliferation, death and survival. Ascytostatic agents, their monotherapy regimen often lacks sufficientclinical benefit. Synergistic outcomes are often obtained by combinationwith cytotoxic agents but are limited by their cumulative side effects.

Most cancer treatments directly or indirectly cause DNA damage in thetreated proliferating tumor cells, which ultimately leads to theirdeath. However, several intrinsic and acquired resistances of tumors tothese treatments are, at least in part, due to the tumor cells'efficient DNA repair activities. It is now well recognized that DNArepair is an important target for cancer therapy (Helleday et al. Nat.Rev. Cancer, 2008, 8:193-204). The most advanced drug development inthis field is the inhibitors of PARP.

As DNA repair is an essential survival process across all livingkingdom, it has multiple specialized repair pathways, and has someredundancies that make the process robust when one pathway is deficientor blocked by a therapeutic agent, such as a DNA repair inhibitor.Therefore, instead of targeting a key gene/protein involved in the DNArepair process, whatever its biological importance and clinicalrelevance, the innovative molecular therapy must deal with one orseveral key pathways as a global target, in conjunction withconventional therapies, so as to reach the most efficient cancertreatment.

It was conceived to globally target DNA lesions' sensing, signaling andrepair pathways in order to disable cancers' defense to existingtreatments. One strategy consists of introducing short modified DNAmolecules mimicking double strand breaks (DSB), named Dbait, into cellsthat until then could efficiently repair DSB and thus survive. Theantitumor efficacy of Dbait in association with radiotherapy (RT) orchemotherapy (CT) is explained by the fact that Dbait molecules trap theinitial DSB sensing complexes, jam downstream repair signaling,subsequently disorganize all DSB repair systems (both Non-Homologous EndJoining and Homologous Recombination pathways), and finally inhibit DSBrepair (WO2005/040378; WO2008/034866; Quanz et al., 2009, ClinicalCancer Research 15:1308; Quanz et al., 2009, PLoS ONE 4:e6298; Dutreixet al., 2010, Mut. Res. 704:182). Ultimately, the cancer cells can nomore escape their death. Dbait molecules have also been found to beeffective alone, without combination with radiotherapy (RT) orchemotherapy (CT) (WO2008/084087).

However, once active agents of clinical interest are identified, therecurrent problem is to find the best way to deliver the active agents,especially for nucleic acid agents. The development and optimization ofefficient non-viral DNA/RNA delivery systems has to address the toxicityissues; the “tissue and systemic barriers” such as degradation,opsonization of particles by charged serum components, rapid clearingand accumulation in non-target tissues when the active substance isadministered by systemic route; and the “cellular barriers” to theirdelivery such as low uptake across the cytoplasmic membrane, inadequaterelease of DNA molecules in the active cellular compartments, and lackof nuclear targeting (required for gene therapy).

Indeed, to be effective, most of these active agents have to be taken upby the cells and to reach the cytoplasm and/or nucleus. In particular,when active agents including nucleic acids are administered in their“free” or naked form, they frequently suffer from degradation before andafter uptake by target cells. Inside the cells, this degradation ismainly due to the fact that nucleic acids enter cells by endocytosis,and are sequestered in cellular endosomes that ultimately evolve intothe lysosomes where chemical and enzymatic degradation is veryefficient.

In the prior art, active agents have been conjugated to various carriersand have been encapsulated into liposomes, micelles and nanoparticleswhere they are protected from degradation in serum. The prior art alsoemploys a variety of chemistries for covalent coupling of nucleic acidsand other active agents to molecular carriers that include polymers suchas dextrans or PEG or molecules aiming to decrease clearance, carriersincluding transferrin, and lipophilic molecules such as cholesterollinked to siRNA to enhance cellular uptake (Chen et al., 2010, J.Controlled Release 144:227). Such carriers may include targetingmoieties such as antibodies, polypeptides, nucleic acids and othersubstances to direct the active agents to selected target cells. Theprior art also discloses molecules improving endocytosis for use inpharmaceutical composition (US2008/0194540).

However, when active DNA/RNA are agents uptaken by cells through theendocytosis process, they frequently end up sequestered in endosomeswhere they are unable to escape, therefore greatly reducing theirtherapeutic potential. For instance, Zimmermann et al. showed that acholesterol-siRNA (ApoB-1) conjugate is about 1000-fold less potent thanits liposomal formulation (SNALP vector) in mice: 100 mg/kg ofcho1-siApoB-1 is equivalent to 0.1 mg/kg of SNALP-ApoB-1 (Zimmermann etal., Nature, 2006, 441:111-114, supplementary FIG. 1).

For nucleic acids, the prior art has tried to solve this problem throughthe use of cationic polymers such as polyethylenimine (PEI)(WO96/02655), liposomes with fusogenic lipids or peptides such as SNALPvector. PEI is able to destabilize endosomes by a well-described protonsponge effect and therefore facilitate the release of the nucleic acid.However, the use of PEI is often limited by its cytotoxicity and so farhas not been approved for use in humans. Liposomal formulation alsoexhibits toxicity and limited encapsulation of nucleic acids (usually inthe range of 1-2 mg/mL) that may not be suitable for the applicationthat requires high payload of nucleic acid agents.

“Endosomolytic” agents such as chloroquines are known to enhance thetransfection of nucleic acids by facilitating their escape fromendosomes into the cytoplasma in cultured cells. However, the use ofchloroquine is limited to in vitro use and has only rarely beenevaluated for assisting nucleic acid delivery in vivo. This may be dueto reports in the art at nucleic acids that teach away from its in vivouse due to chloroquine toxicity.

Benns, et al. (2000, Bioconj. Chem. 11: 637) reported that “Althoughchloroquine has proven to aid in the release of the plasma DNA into thecytoplasm, it has been found to be toxic and thus cannot be used invivo”. This problem is partly due to the fact that relatively highconcentrations of free chloroquine are needed to reach the same site asthe nucleic acid (i.e., plasmid DNA) in the endosome. Similarly, Zhanget al. (2003, Gene Med 5:209) studied in vivo use of chloroquine forgene delivery to the liver. In this article, they used a plasmidtogether with a peptide (polylysine/molossin) as a DNA vector. Theyconcluded that, despite chloroquine being effective for promoting genedelivery to the liver, multiple dosing is required and its use islimited by systemic toxicity. Indeed, they demonstrated that the acutesystemic chloroquine toxicity limits in vivo use to levels which aresubstantially below those required for optimal gene delivery. Localdelivery of chloroquine is also limited by local toxicity of chloroquineand by its diffusion away from the site of delivery. Finally, they didnot observe a gene delivery, or a very low level, when naked DNA isused.

In this context, WO2007/040469 discloses that the solution to theproblem of the high necessary concentration of chloroquine may beovercome by covalently coupling the chloroquine to the active agent,thereby reducing the overall dosage needed. WO2009/126933 proposes tocovalently link the nucleic acid to deliver both to an endosomolyticagent and to a targeting ligand.

Chloroquine and its derivatives such as hydroxychloroquine are used incurative and prophylactic treatment of malaria. It has also been studiedfor use in combination with radiotherapy and/or chemotherapy of cancers(Sotelo et al., 2006, Ann Intern Med 144:337-342; NCT01023477 andNCT00969306). The hypothesis is that chloroquine/hydroxychloroquineinhibits autophagy, which is a normal cell defense process, by exportingtherapeutic agents to lysosomes where they are degraded.

In conclusion, optimization of nucleic acid-based therapies requiresfurther addressing the efficiency and cytotoxicity of synthetic DNAdelivery systems.

SUMMARY OF THE INVENTION

The present invention provides a new efficient method for in vivodelivery of nucleic acids of therapeutic interest based on the covalentconjugation of nucleic acids of therapeutic interest with a moleculefacilitating endocytosis, and the use of conjugated nucleic acids oftherapeutic interest in combination with an endosomolytic agent. Inparticular, this in vivo delivery system is used for Dbait molecules.

Therefore, the present invention relates to a pharmaceutical compositioncomprising a conjugated nucleic acid molecule having at least one freeend and a DNA double-stranded portion of 20-200 bp with less than 60%sequence identity to any gene in a human genome, said nucleic acidmolecule being covalently linked to a molecule facilitating endocytosisselected from a lipophilic molecule or a ligand which targets cellreceptors enabling receptor-mediated endocytosis, and a quinolineendosomolytic agent. The pharmaceutical composition may further comprisea DNA-damaging antitumoral agent.

The present invention also relates to a product comprising a conjugatednucleic acid molecule having at least one free end and a DNAdouble-stranded portion of 20-200 bp with less than 60% sequenceidentity to any gene in a human genome, said nucleic acid molecule beingcovalently linked to a molecule facilitating endocytosis selected from alipophilic molecule or a ligand which targets cell receptors enablingreceptor-mediated endocytosis, and a quinoline endosomolytic agent, as acombined preparation for simultaneous, separate or sequential use. Theproduct may further comprise a DNA-damaging antitumoral agent.Preferably, the quinoline endosomolytic agent is to be administeredbefore and/or simultaneously with the conjugated nucleic acid molecule.In particular, the quinoline endosomolytic agent is to be administeredas a pre-treatment of at least one week by the oral route, and then theconjugated nucleic acid molecule and the quinoline endosomolytic agentare to be administered as a combined preparation for simultaneous,separate or sequential use.

The present invention relates to the pharmaceutical composition orproduct as disclosed herein for use in the treatment of cancer.Preferably, the treatment further comprises radiotherapy orchemotherapy, preferably with a DNA-damaging antitumoral agent.Optionally, the DNA-damaging antitumoral agent is selected from thegroup consisting of an inhibitor of topoisomerases I or II, a DNAcrosslinker, a DNA alkylating agent, an anti-metabolic agent andinhibitors of the mitotic spindles.

In a preferred embodiment, the quinoline endosomolytic agent ischloroquine or hydroxychloroquine, preferably chloroquine.

In a preferred embodiment, the molecule facilitating endocytosis isselected from the group consisting of single or double chain fatty acidssuch as octodecyl and dioleoyl, tocopherol, folates or folic acid,cholesterol, sugars such as galactose and mannose and theiroligosaccharides, peptides such as RGD and bombesin, and proteins suchas integrin, preferably single or double chain fatty acids, folates andcholesterol, more preferably dioleoyl, octadecyl, folic acid, andcholesterol, still more preferably cholesterol.

In a preferred embodiment, the conjugated nucleic acid molecule has oneof the following formulae:

wherein N is a nucleotide, n is an integer from 15 to 195, theunderlined N refers to a nucleotide having or not having a modifiedphosphodiester backbone, L′ is a linker, C is the molecule facilitatingendocytosis, L is a linker, and m is an integer being 0 or 1.Preferably, the underlined N refers to a nucleotide having a modifiedphosphodiester backbone. Preferably, the linker L′ is selected from thegroup consisting of hexaethyleneglycol, tetradeoxythymidylate (T4) and2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane; and/or mis 1 and L is a carboxamido oligoethylene glycol, preferably acarboxamido triethylene glycol; and/or C is selected from the groupconsisting of single or double chain fatty acids, folates andcholesterol. More preferably, the linker L′ is selected from the groupconsisting of hexaethyleneglycol, tetradeoxythymidylate (T4) and2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane; and m is1 and L is a carboxamido oligoethylene glycol, preferably a carboxamidotriethylene glycol; and C is selected from the group consisting ofsingle or double chain fatty acids, tocopherol, folates or folic acid,cholesterol, sugars such as galactose and mannose and theiroligosaccharides, peptides such as RGD and bombesin, and proteins suchas integrin, preferably single or double chain fatty acids, folates andcholesterol. More preferably, C is selected from the group consisting ofdioleoyl, octadecyl, folic acid, and cholesterol. Still more preferablyC is cholesterol.

In a more particular embodiment, the conjugated nucleic acid moleculehas one of the following formulae:

wherein the underlined nucleotide refers to a nucleotide having or nothaving a phosphorothioate or methylphosphonate backbone, L′ is a linker,C is the molecule facilitating endocytosis, L is a linker, and m is aninteger being 0 or 1. Preferably, the underlined nucleotide refers to anucleotide have a phosphorothioate or methylphosphonate backbone.Preferably, the underlined nucleotide refers to a nucleotide having aphosphorothioate backbone; and/or the linker L′ is selected from thegroup consisting of hexaethyleneglycol, tetradeoxythymidylate (T4) and2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane; and/or mis 1 and L is a oligoethylene glycol, preferably a carboxamidotriethyleneglycol, carboxamido tetraethyleneglycol, or carboxamidooligoethylene glycol, more preferably a carboxamido triethylene glycol;and/or C is selected from the group consisting of single or double chainfatty acids, tocopherol, folates or folic acid, cholesterol, sugars suchas galactose and mannose and their oligosaccharides, peptides such asRGD and bombesin, and proteins such as integrin, preferably single ordouble chain fatty acids, folates and cholesterol. More preferably, thelinker L′ is selected from the group consisting of hexaethyleneglycol,tetradeoxythymidylate (T4) and2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane; and m is1 and L is a carboxamido polyethylene glycol, preferably a carboxamidotriethylene glycol; and C is selected from the group consisting ofsingle or double chain fatty acids, folates and cholesterol. Morepreferably, C is selected from the group consisting of dioleoyl,octadecyl, folic acid, and cholesterol. Still more preferably, C ischolesterol.

In a very specific embodiment, the conjugated nucleic acid molecule is

wherein the underlined nucleotide refers to a nucleotide having aphosphorothioate backbone.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication, withcolor drawing(s), will be provided by the Office upon request andpayment of the necessary fee.

FIGS. 1A-1B: Cellular uptake of formulated Dbait. (A) Microscopyanalysis of Dbait complexes with PEI11k (a). (B) Flow cytometry analysisof cellular uptake was performed 5 hours after beginning of treatmentfor various transfection conditions. Dbait-cy3 with SuperFect, 2 μg/mlcoDbait-cy3 without and with chloroquine treatment prior transfection,25 μg/ml coDbait-cy3 without and with CQ.

FIGS. 2A-2C: Activity of formulated Dbait. (A) DNA-PK activation wasmeasured after addition of 50 u purified enzyme complex to no DNA, 0.25μg Dbait or 0.25 μg coDbait. (B) Immunodetection of γ-H2AX in cells 24hours after treatment with 1.6 μg/ml Dbait (left), 1.6 μg/mlDbait/PEI11K (middle), 16 μg/ml coDbait with CQ. Scale bar: 20 μm. (C)Quantification of γ-H2AX, 5 hours (black) and 24 hours (grey) aftertreatment by various formulated Dbait. All transfections were performedwith 1.6 μg/mL Dbait or 16 μg/mL coDbait. When indicated, CQ was addedprior to transfection.

FIGS. 3A-3D: Phenotypes 24 hours after Dbait injection into theextracellular space of cell stage 1K zebrafish embryos. (A-C) Lateralviews anterior to the left of zebrafish embryos 24 hours afterDbait-cy3+PEI injection (2-5 nL) at the animal pole of cell stage 1Kzebrafish embryos: top panel, bright field view; bottom panel, 2×magnification of the head region with epifluorescence overlay showing inred Dbait-cy3. (A) Type 1 phenotype undistinguishable from non-injected(not shown). (B) Type 2 mild phenotype with extensive cell death in thehead region. (C) Type 3 strong teratogenesis and widespread cell death.(D) Histogram showing the percentage of the three phenotypic classesdepending on the adjuvant. More than 100 embryos were analyzed for eachcondition. NA: Dbait injected alone; Sup: SuperFect; 25 k, 22 k, 11 kPEI of the corresponding size; chloro: chloroquine; Lut: Lutrol.

FIG. 4: Diffusion and activity in tumors. Tumors were injected with 1.6μg Dbait-cy5.5/PEI or 16 μg coDbait-cy5.5 ( 1/10 of cy5.5 labeledcoDbait+9/10 unlabeled coDbait in order to keep similar fluorescenceintensity) and analyzed the following day for fluorescence distributionand DNA-PKcs activity. Diffusion of fluorescent Dbait after two types ofinjections: one intratumoral injection or two subcutaneous injections.

FIG. 5: Survival of 5 groups of nude mice bearing SK28 melanomaxenograft: 1) untreated (n=16); 2) irradiated (IR, n=12); 3) irradiatedwith intraperitoneally injected 1 mg chloroquine (CQ, IR, n=10); 4)treated by intratumorally injected 0.6 mg DT01 (also called CoDbait) andirradiated 5 hours later (DT01, IR, n=11) and 5) pretreated withintraperitoneally injected 1 mg chloroquine 2 hours prior intratumoralinjection of 0.6 mg DT01 (also called CoDbait) and irradiated 5 hourslater (DT01, CQ, IR, n=13).

FIG. 6. Study of tumor growth of melanoma SK28 grafted onto nude mice.Top: treatment protocol: 4 treatments of DT01 (also called CoDbait)combined with 4 irradiation (RT) sessions in two weeks. 4 mg DT01 weresubcutaneously injected at two opposite points separated by 5 mm fromthe tumor border. Animals were pre-treated before beginning of treatmentand during the DT01+RT treatment with 1 mg chloroquine (CQ) twice a weekby oral administration (p.o.)*. Middle: Mean value of tumor growth ofvarious animal groups; Untreated or CQ: untreated or only treated by CQ(n=11); RT or CQ+RT: irradiated with or without a co-treatment ofchloroquine (n=16); DT01+RT: treated by DT01 and irradiation (n=10);DT01+CQ+RT: treated by DT01 with chloroquine and irradiation (n=12).Bottom: Details of groups DT01+RT and DT01+CQ+RT. Each curve correspondsto one tumor growth.

FIG. 7: DNA-PK activity was monitored using the SignaTECT DNA-dependentProtein Kinase Assay System kit (Promega, Madison, Wis., USA). Thebiotinylated peptide substrate, 50 units of DNA-PK (Promega, Madison,Wis., USA) and 500 nM various Dbait molecules were incubated for 5 minat 30° C. with (γ-32P) ATP according to the manufacturer's instructions.The biotinylated substrate was captured on a streptavidin membrane,washed and counted in a scintillation counter. Percentage ofphosphorylation was calculated by dividing the bound radioactivity bythe total count of (γ-³²P) ATP per sample. Dbait32Hc is an unconjugatedDbait molecule. 0813, 0815, 0902, 0903, 0904 and 0905 are conjugatedDbait molecules (cf. Tables of “Alternative conjugated Dbaitmolecules”). Dbait8H is a short (8-bp) Dbait used as a negative controlof DNA-PK activity.

FIG. 8: Activity of Dbait molecules measured by H2AX phosphorylation.Immunodetection of the γ-H2AX in MRC5 cell line 24 hours after thetransfection of various conjugated Dbait molecules (cf. Tables of“Alternative conjugated Dbait molecules”) with or without priortreatment of 50 μM chloroquine. Dbait formulated by polyethylenimine(PEI) was used as a positive control.

DETAILED DESCRIPTION OF THE INVENTION

Introducing small DNA molecules (Dbait) impairs DNA repair of damagedchromosomes and provides an efficient method for enhancing radiotherapyor chemotherapy efficiency in tumors, in particular in resistant tumors.However, the sensitizing activity of the Dbait molecules depends uponthe efficiency of their delivery within the tumoral cells.

Therefore, the inventors compared different strategies to improve thiskey step. In order to test the strategies, they developed a pipeline ofassays: (i) molecular analysis of complexes formed with Dbait molecules,(ii) cellular tests for Dbait uptake and activity, and (iii) livezebrafish embryo confocal microscopy monitoring for in vivo distributionand biological activity of the formulated molecules. These tests allowedthe selection of the most efficient formulations and administrationprotocols before assays on xenografted tumours on mice. Two classes offormulation were compared: polycationic polymers with linear or branchedpolyethylenimine (PEI), and Dbait covalently attached cholesterol(coDbait). The linear PEI complexes were the most efficient for Dbaittransfection in vitro and in vivo but displayed high toxicity. Indeed,10-fold higher doses of coDbait used with 1 mg chloroquine (according toallometric conversion, equivalent to the prophylactic dose used inhumans) were required to observe the same antitumoral effect onxenografted melanomas than Dbait when formulated with PEI. However, thetested doses of coDbait administered with chloroquine were found to benon-toxic.

Accordingly, the present invention describes the combination and theadministration protocol of a cholesterol-nucleic acid conjugate withsystemically administered chloroquine at clinically relevant doses, inparticular the use of a cholesterol-Dbait conjugate with a prophylacticdose of chloroquine (by allometric conversion into animals), withoutnotable toxicity. The inventors showed that a 10-fold higher amount(10×) of cholesterol-Dbait has similar potency as compared to 1× Dbaitvectorized by a non-viral vector system in mice, instead of 1000-foldwithout the use of chloroquine as described in the prior art. This makesthe conjugation of therapeutic nucleic acids to a lipophilic orcell-targeting agent a safe and economically usable delivery system.

Accordingly, despite the need of a higher dose of coDbait, the inventorssurprisingly found that:

-   -   1) The combination of coDbait with chloroquine presents low, if        any, toxicity in vivo. It allows to improve the therapeutic        index (ratio of efficacy dose/toxicity dose) from almost 1 for        Dbait/PEI to >20 for coDbait; the absence of toxicity has been        observed after intravenous injection, subcutaneous injection and        even after intracerebral injection in mouse, rat, rabbit and        monkey.    -   2) The combination of coDbait with chloroquine gives a retarded        and sustained activation of DNA-PK (the main target of Dbait)        and allows a prolonged therapeutic effect. More particularly, an        increasing activity or effect is observed through the time        period.    -   3) Surprisingly, the coDbait is well diffused in tumor/tissue        compared with Dbait/PEI.    -   4) For the first time, the inventors observed that chloroquine        allows the cellular uptake increase of coDbait, whereas this        effect is less pronounced with cholesterol conjugated to siRNA        molecules.        Based on these observations, the present invention relates to:    -   a pharmaceutical composition comprising a) a conjugated Dbait        molecule or hairpin nucleic acid molecule as described below,        and optionally b) a DNA-damaging anti-tumoral agent, and a        pharmaceutically acceptable carrier, in particular for use in        the treatment of cancer;    -   a pharmaceutical composition comprising a) a conjugated Dbait        molecule or hairpin nucleic acid molecule as described below, b)        an endosomolytic agent as described below, and optionally c) a        DNA-damaging anti-tumoral agent, and a pharmaceutically        acceptable carrier, in particular for use in the treatment of        cancer;    -   a product or kit containing a) a conjugated Dbait molecule or        hairpin nucleic acid molecule as disclosed below, and        optionally b) a DNA-damaging anti-tumoral agent, as a combined        preparation for simultaneous, separate or sequential use, in        particular in the treatment of cancer;    -   a product or kit containing a) a conjugated Dbait molecule or        hairpin nucleic acid molecule as disclosed below, b) an        endosomolytic agent as described below, and optionally c) a        DNA-damaging anti-tumoral agent, as a combined preparation for        simultaneous, separate or sequential use, in particular in the        treatment of cancer;    -   a combined preparation which comprises a) a conjugated Dbait        molecule or hairpin nucleic acid molecule as disclosed below, b)        an endosomolytic agent as described below, and optionally c) a        DNA-damaging anti-tumoral agent for simultaneous, separate or        sequential use, in particular in the treatment of cancer;    -   a pharmaceutical composition comprising a conjugated Dbait        molecule or hairpin nucleic acid molecule as disclosed below,        for the use in the treatment of cancer in combination with        radiotherapy and/or a DNA-damaging anti-tumoral agent;    -   a pharmaceutical composition comprising a) a conjugated Dbait        molecule or hairpin nucleic acid molecule as disclosed below,        and b) an endosomolytic agent as described below, for the use in        the treatment of cancer in combination with radiotherapy and/or        a DNA-damaging anti-tumoral agent;    -   a product or kit containing a) a conjugated Dbait molecule or        hairpin nucleic acid molecule as disclosed below, and b) an        endosomolytic agent as described below, as a combined        preparation for simultaneous, separate or sequential use, in        particular in the treatment of cancer in combination with        radiotherapy and/or a DNA-damaging anti-tumoral agent;    -   the use of a pharmaceutical composition comprising a conjugated        Dbait molecule or hairpin nucleic acid molecule as disclosed        below for the manufacture of a medicament for the treatment of        cancer in combination with radiotherapy and/or a DNA-damaging        anti-tumoral agent or for increasing the efficiency of the        treatment of cancer with radiotherapy and/or a DNA-damaging        anti-tumoral agent or for enhancing tumor sensitivity to        radiotherapy and/or to treatment with a DNA-damaging        anti-tumoral agent;    -   the use of a pharmaceutical composition comprising a conjugated        Dbait molecule or hairpin nucleic acid molecule as disclosed        below for the manufacture of a medicament for the treatment of        cancer in combination with radiotherapy and/or a DNA-damaging        anti-tumoral agent and with an endosomolytic agent as disclosed        below;    -   the use of a pharmaceutical composition comprising a) a        conjugated Dbait molecule or hairpin nucleic acid molecule as        disclosed herein, and b) an endosomolytic agent as described        below for the manufacture of a medicament for the treatment of        cancer in combination with radiotherapy and/or a DNA-damaging        anti-tumoral agent;    -   the use of a pharmaceutical composition comprising a) a        conjugated Dbait molecule or hairpin nucleic acid molecule as        disclosed herein, and b) an endosomolytic agent as described        below for the manufacture of a medicament for increasing the        efficiency of the treatment of cancer with radiotherapy and/or a        DNA-damaging anti-tumoral agent or for enhancing tumor        sensitivity to radiotherapy and/or to treatment with a        DNA-damaging anti-tumoral agent;    -   the use of a pharmaceutical composition comprising a) a        conjugated Dbait molecule or hairpin nucleic acid molecule as        disclosed herein, b) an endosomolytic agent as described below,        and optionally c) a DNA-damaging anti-tumoral agent and a        pharmaceutically acceptable carrier for the manufacture of a        medicament for the treatment of cancer;    -   a method for treating a cancer in a subject in need thereof,        comprising administering an effective amount of a pharmaceutical        composition comprising a conjugated Dbait molecule or hairpin        nucleic acid molecule as disclosed herein, and optionally a        DNA-damaging anti-tumoral agent, and a pharmaceutically        acceptable carrier;    -   a method for treating a cancer in a subject in need thereof,        comprising administering an effective amount of a pharmaceutical        composition comprising a) a conjugated Dbait molecule or hairpin        nucleic acid molecule as disclosed herein, b) an endosomolytic        agent as described below, and optionally c) a DNA-damaging        anti-tumoral agent, and a pharmaceutically acceptable carrier;    -   a method for treating a cancer in a subject in need thereof,        comprising administering an effective amount of a pharmaceutical        composition comprising a conjugated Dbait molecule or hairpin        nucleic acid molecule as disclosed herein, an effective amount        of a pharmaceutical composition comprising an endosomolytic        agent as described below and optionally an effective amount of a        pharmaceutical composition comprising a DNA-damaging        anti-tumoral agent;    -   a method for treating a cancer in a subject in need thereof,        comprising administering an effective amount of a pharmaceutical        composition comprising a) a conjugated Dbait molecule or hairpin        nucleic acid molecule as disclosed herein and b) an effective        amount of a pharmaceutical composition comprising an        endosomolytic agent as described below in combination with        radiotherapy and/or a DNA-damaging anti-tumoral agent;    -   a method for increasing the efficiency of a treatment of a        cancer with radiotherapy and/or a DNA-damaging anti-tumoral        agent or for enhancing tumor sensitivity to radiotherapy and/or        to treatment with a DNA-damaging anti-tumoral agent in a subject        in need thereof, comprising administering an effective amount of        a pharmaceutical composition comprising a) a conjugated Dbait        molecule or hairpin nucleic acid molecule as disclosed herein        and b) an endosomolytic agent as described below, and a        pharmaceutically acceptable carrier; and    -   a method for increasing the efficiency of a treatment of a        cancer with radiotherapy and/or a DNA-damaging anti-tumoral        agent or for enhancing tumor sensitivity to radiotherapy and/or        to treatment with a DNA-damaging anti-tumoral agent in a subject        in need thereof, comprising administering an effective amount of        a pharmaceutical composition comprising a conjugated Dbait        molecule or hairpin nucleic acid molecule as disclosed herein,        and an effective amount of a pharmaceutical composition        comprising an endosomolytic agent as described below.

The terms “kit”, “product” or “combined preparation”, as used herein,define especially a “kit of parts” in the sense that the combinationpartners (a) and (b), and optionally (c), as defined above can be dosedindependently or by use of different fixed combinations withdistinguished amounts of the combination partners (a) and (b), andoptionally (c), i.e., simultaneously or at different time points. Theparts of the kit of parts can then, e.g., be administered simultaneouslyor chronologically staggered, that is at different time points and withequal or different time intervals for any part of the kit of parts. Theratio of the total amounts of the combination partner (a) to thecombination partner (b), and optionally (c), to be administered in thecombined preparation can be varied. The combination partners (a) and(b), and optionally (c), can be administered by the same route or bydifferent routes.

Within the context of the invention, the term “treatment” denotescurative, symptomatic, and preventive treatment. Pharmaceuticalcompositions, kits, products and combined preparations of the inventioncan be used in humans with an existing cancer or tumor, including atearly or late stages of progression of the cancer. The pharmaceuticalcompositions, kits, products and combined preparations of the inventionwill not necessarily cure the patient who has the cancer but will delayor slow the progression or prevent further progression of the disease,improving thereby the patient's condition. In particular, thepharmaceutical compositions, kits, products and combined preparations ofthe invention reduce the development of tumors, reduce tumor burden,produce tumor regression in a mammalian host and/or prevent metastasisoccurrence and cancer relapse. In treating the cancer, thepharmaceutical composition of the invention is administered in atherapeutically effective amount.

By “effective amount” it is meant the quantity of the pharmaceuticalcomposition of the invention which prevents, removes or reduces thedeleterious effects of cancer in mammals, including humans, alone or incombination with the other active ingredients of the pharmaceuticalcomposition, kit, product or combined preparation. It is understood thatthe administered dose may be adapted by those skilled in the artaccording to the patient, the pathology, the mode of administration,etc.

Whenever the term “treatment of a cancer” or the like is used withinthis specification with reference to the pharmaceutical composition ofthe invention, it refers to: a) a method for treating a cancer, saidmethod comprising administering a pharmaceutical composition of theinvention to a subject in need of such treatment; b) the use of apharmaceutical composition of the invention for the treatment of acancer; c) the use of a pharmaceutical composition of the invention forthe manufacture of a medicament for the treatment of a cancer; and/or d)a pharmaceutical composition of the invention for use in the treatment acancer.

Dbait Molecules

Dbait molecules have been extensively described in PCT patentapplications WO2005/040378, WO2008/034866 and WO2008/084087, thedisclosures of which are incorporated herein by reference.

Dbait molecules may be defined by a number of characteristics necessaryfor their therapeutic activity, such as their minimal length, thepresence of at least one free end, and the presence of a double-strandedportion, preferably a double-stranded DNA portion. As will be discussedbelow, it is important to note that the precise nucleotide sequence ofDbait molecules does not impact their activity. Furthermore, Dbaitmolecules may contain a modified and/or non-natural backbone.

Preferably, Dbait molecules are of non-human origin (i.e., theirnucleotide sequence and/or conformation (e.g., hairpin) does not existas such in a human cell), most preferably of synthetic origin. As thesequence of the Dbait molecules plays little, if any, role, Dbaitmolecules have preferably no significant degree of sequence homology oridentity to known genes, promoters, enhancers, 5′- or 3′-upstreamsequences, exons, introns, and the like. In other words, Dbait moleculeshave less than 80% or 70%, even less than 60% or 50% sequence identityto any gene in a human genome. Methods of determining sequence identityare well known in the art and include, e.g., BLASTN 2.2.25. By humangenome, it is preferably considered for determining the identitypercentage the Human Genome Build 37 (reference GRCh37.p2 and alternateassemblies). Dbait molecules do not hybridize, under stringentconditions, with human genomic DNA. Typical stringent conditions aresuch that they allow the discrimination of fully complementary nucleicacids from partially complementary nucleic acids.

In addition, the sequence of the Dbait molecules is preferably devoid ofCpG in order to avoid the well-known toll-like receptor-mediatedimmunological reactions.

The length of Dbait molecules may be variable, as long as it issufficient to allow appropriate binding of Ku protein complexescomprising Ku and DNA-PKcs proteins. It has been shown that the lengthof Dbait molecules must be greater than 20 bp, preferably about 32 bp,to ensure binding to such a Ku complex and allow DNA-PKcs activation.Preferably, Dbait molecules comprise between 20-200 bp, more preferably24-100 bp, still more preferably 26-100, and most preferably between32-100 bp. For instance, Dbait molecules comprise between 24-160,26-150, 28-140, 30-120, or 32-100 bp. By “bp” is intended that themolecule comprise a double stranded portion of the indicated length.

In a particular embodiment, the Dbait molecules having a double-strandedportion of at least 32 bp, or of about 32 bp, comprise the samenucleotide sequence as Dbait32 (SEQ ID NO: 1), Dbait32Ha (SEQ ID NO: 2),Dbait32Hb (SEQ ID NO: 3), Dbait32Hc (SEQ ID NO: 4) or Dbait32Hd (SEQ IDNO: 5). Optionally, the Dbait molecules have the same nucleotidecomposition as Dbait32, Dbait32Ha, Dbait32Hb, Dbait32Hc or Dbait32Hd buttheir nucleotide sequence is different; then the Dbait moleculescomprise one strand of the double-stranded portion with 3 A, 6 C, 12 Gand 11 T. Preferably, the sequence of the Dbait molecules does notcontain any CpG dinucleotide.

Alternatively, the double-stranded portion comprises at least 16, 18,20, 22, 24, 26, 28, 30 or 32 consecutive nucleotides of Dbait32 (SEQ IDNO: 1), Dbait32Ha (SEQ ID NO: 2), Dbait32Hb (SEQ ID NO: 3), Dbait32Hc(SEQ ID NO: 4) or Dbait32Hd (SEQ ID NO: 5). In a more particularembodiment, the double-stranded portion consists of 20, 22, 24, 26, 28,30 or 32 consecutive nucleotides of Dbait32 (SEQ ID NO: 1), Dbait32Ha(SEQ ID NO: 2), Dbait32Hb (SEQ ID NO: 3), Dbait32Hc (SEQ ID NO: 4) orDbait32Hd (SEQ ID NO: 5).

Dbait must have at least one free end, as a mimic of DSB. Said free endmay be either a free blunt end or a 5′-/3′-protruding end. The “freeend” refers herein to a nucleic acid molecule, in particular adouble-stranded nucleic acid portion, having both a 5′ end and a 3′ endor having either a 3′ end or a 5′ end. Optionally, one of the 5′ and 3′end can be used to conjugate the Dbait molecule or can be linked to ablocking group, for instance a 3′-3′ nucleotide linkage.

In a particular embodiment, they contain two free ends and can belinear. Accordingly, the Dbait molecules may also be double-strandedmolecules with two free ends and having the nucleotide sequence ofDbait32 (SEQ ID NO: 1), Dbait32Ha (SEQ ID NO: 2), Dbait32Hb (SEQ ID NO:3), Dbait32Hc (SEQ ID NO: 4) or Dbait32Hd (SEQ ID NO: 5).

In another particular embodiment, they contain only one free end.Preferably, the Dbait molecules are made of hairpin nucleic acids with adouble-stranded DNA stem and a loop. The loop can be a nucleic acid orother chemical groups known by a skilled person or a mixture thereof. Anucleotide linker may include from 2 to 10 nucleotides, preferably 3, 4or 5 nucleotides. Non-nucleotide linkers non-exhaustively include abasicnucleotides, polyethers, polyamines, polyamides, peptides,carbohydrates, lipids, polyhydrorarbons, or other polymeric compounds(e.g., oligoethylene glycols such as those having between 2 and 10ethylene glycol units, preferably 4, 5, 6, 7 or 8 ethylene glycolunits). A preferred linker is selected from the group consisting ofhexaethyleneglycol, tetradeoxythymidylate (T4) and other linkers such as2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane.Accordingly, in a particular embodiment, the Dbait molecules can behairpin molecules having a double-stranded portion or stem comprising atleast 16, 18, 20, 22, 24, 26, 28, 30 or 32 consecutive nucleotides ofDbait32 (SEQ ID NO: 1), Dbait32Ha (SEQ ID NO: 2), Dbait32Hb (SEQ ID NO:3), Dbait32Hc (SEQ ID NO: 4) or Dbait32Hd (SEQ ID NO: 5) and a loopbeing a hexaethyleneglycol linker, a tetradeoxythymidylate linker (T4)or 2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane. In amore particular embodiment, those Dbait molecules can have adouble-stranded portion consisting of 20, 22, 24, 26, 28, 30 or 32consecutive nucleotides of Dbait32 (SEQ ID NO: 1), Dbait32Ha (SEQ ID NO:2), Dbait32Hb (SEQ ID NO: 3), Dbait32Hc (SEQ ID NO: 4) or Dbait32Hd (SEQID NO: 5).

Dbait molecules preferably comprise a 2′-deoxynucleotide backbone, andoptionally comprise one or several (2, 3, 4, 5 or 6) modifiednucleotides and/or nucleobases other than adenine, cytosine, guanine andthymine. Accordingly, the Dbait molecules are essentially a DNAstructure. In particular, the double-stranded portion or stem of theDbait molecules is made of deoxyribonucleotides.

Preferred Dbait molecules comprise one or several chemically modifiednucleotide(s) or group(s) at the end of one or each strand, inparticular in order to protect them from degradation. In a particularpreferred embodiment, the free end(s) of the Dbait molecules is(are)protected by one, two or three modified phosphodiester backbones at theend of one or each strand. Preferred chemical groups, in particular themodified phosphodiester backbone, comprise phosphorothioates.Alternatively, preferred Dbait have 3′-3′ nucleotide linkage, ornucleotides with methylphosphonate backbones. Other modified backbonesare well known in the art and comprise phosphoramidates, morpholinonucleic acid, 2′-0,4′-C methylene/ethylene bridged locked nucleic acid,peptide nucleic acid (PNA), and short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintrasugar linkages of variable length, or any modified nucleotidesknown by a skilled person. In a first preferred embodiment, the Dbaitmolecules have the free end(s) protected by one, two or three modifiedphosphodiester backbones at the end of one or each strand, morepreferably by three modified phosphodiester backbones (in particularphosphorothioate or methylphosphonate) at least at the 3′end, but stillmore preferably at both the 5′ and 3′ ends.

In a most preferred embodiment, the Dbait molecule is a hairpin nucleicacid molecule comprising a DNA double-stranded portion or stem of 32 bp(e.g., with a sequence selected from the group consisting of SEQ ID NOS:1-5, in particular SEQ ID NO: 4) and a loop linking the two strands ofthe DNA double-stranded portion or stem comprising or consisting of alinker selected from the group consisting of hexaethyleneglycol,tetradeoxythymidylate (T4) and2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane, the freeends of the DNA double-stranded portion or stem (i.e., at the oppositeof the loop) having three modified phosphodiester backbones (inparticular phosphorothioate internucleotidic links).

Said Dbait molecules are made by chemical synthesis, semi-biosynthesisor biosynthesis, any method of amplification, followed by any extractionand preparation methods and any chemical modification. Linkers areprovided so as to be incorporable by standard nucleic acid chemicalsynthesis.

More preferably, Dbait molecules are manufactured by specially designedconvergent synthesis: two complementary strands are prepared by standardnucleic acid chemical synthesis with the incorporation of an appropriatelinker precursor, and after their purification, they are covalentlycoupled together.

Conjugated Dbait Molecules

The present invention concerns Dbait molecules conjugated to moleculesfacilitating endocytosis or cellular uptake.

In particular, the molecules may be lipophilic molecules such ascholesterol, single or double chain fatty acids, or ligands which targetcell receptors, enabling receptor-mediated endocytosis, such as folicacid and folate derivatives or transferrin (Goldstein et al., Ann. Rev.Cell Biol., 1985, 1:1-39; Leamon & Lowe, Proc Natl Acad Sci USA, 1991,88: 5572-5576). Fatty acids may be saturated or unsaturated and be inC₄-C₂₈, preferably in C₁₄-C₂₂, still more preferably in C₁₈ such asoleic acid or stearic acid. In particular, fatty acids may be octadecylor dioleoyl. Fatty acids may be found in double chain form linked withan appropriate linker such as a glycerol, a phosphatidylcholine orethanolamine and the like or linked together by the linkers used toattach to the Dbait molecule. As used herein, the term “folate” refersto folate and folate derivatives, including pteroic acid derivatives andanalogs. The analogs and derivatives of folic acid suitable for use inthe present invention include, but are not limited to, antifolates,dihydrofolates, tetrahydrofolates, folinic acid, pteropolyglutamic acid,1-deza, 3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10-dideaza,8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroic acidderivatives. Additional folate analogs are described in US2004/242582.The molecule facilitating endocytosis may be tocopherol, a sugar such asgalactose and mannose and their oligosaccharides, a peptide such as RGDand bombesin, or a protein such as integrin. Accordingly, the moleculefacilitating endocytosis may be selected from the group consisting ofsingle or double chain fatty acids, folates and cholesterol. Morepreferably, the molecule facilitating endocytosis is selected from thegroup consisting of dioleoyl, octadecyl, folic acid, and cholesterol. Ina most preferred embodiment, the Dbait molecule is conjugated to acholesterol.

The molecules facilitating endocytosis are conjugated to the Dbaitmolecules, preferably through a linker. Any linker known in the art maybe used to covalently attach the molecule facilitating endocytosis tothe Dbait molecules. For instance, WO09/126933 provides a broad reviewof convenient linkers pages 38-45. The linker can be, non-exhaustively,an aliphatic chain, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, or other polymeric compound (e.g.,oligoethylene glycols such as those having between 2 and 10 ethyleneglycol units, preferably 3, 4, 5, 6, 7 or 8 ethylene glycol units, stillmore preferably 6 ethylene glycol units), as well as incorporating anybonds that may be broken down chemically or enzymatically, such as adisulfide linkage, a protected disulfide linkage, an acid labile linkage(e.g., a hydrazone linkage), an ester linkage, an ortho ester linkage, aphosphonannde linkage, a biocleavable peptide linkage, an azo linkage oran aldehyde linkage. Such cleavable linkers are detailed inWO2007/040469, pages 12-14, and WO2008/022309, pages 22-28.

In a particular embodiment, the Dbait molecule can be linked to onemolecule facilitating endocytosis. Alternatively, several moleculesfacilitating endocytosis (e.g., two, three or four) can be attached toone Dbait molecule.

In a specific embodiment, the linker between the molecule facilitatingendocytosis, in particular cholesterol, and the Dbait molecule isCO—NH—(CH₂—CH₂—O)_(n), wherein n is an integer from 1 to 10, preferablyn being selected from the group consisting of 3, 4, 5 and 6. In a veryparticular embodiment, the linker is CO—N—(CH₂—CH₂—O)₄ (carboxamidotriethylene glycol). The linker can be linked to the Dbait molecules atany convenient position which does not modify the activity of the Dbaitmolecules. In particular, the linker can be linked at the 5′ end, at the3′ end or in the loop when the Dbait molecule is a hairpin. However, inthe case of a hairpin Dbait molecule, the inventors surprisingly foundthat cholesterol linked to the Dbait molecule through a linker at its 5′end is more efficient than cholesterol linked to the Dbait moleculethrough a linker at the loop. Therefore, in a preferred embodiment, thecontemplated conjugated Dbait molecule is a Dbait molecule having ahairpin structure and being conjugated to the molecule facilitatingendocytosis, preferably through a linker, at its 5′ end.

In another specific embodiment, the linker between the moleculefacilitating endocytosis, in particular cholesterol, and the Dbaitmolecule is dialkyl-disulfide (e.g., (CH₂)_(p)—S—S—(CH₂)_(q) with p andq being integer from 1 to 10, preferably from 3 to 8, for instance 6).

In a most preferred embodiment, the conjugated Dbait molecule is ahairpin nucleic acid molecule comprising a DNA double-stranded portionor stem of 32 bp (e.g., with a sequence selected from the groupconsisting of SEQ ID NOS: 1-5, in particular SEQ ID NO: 4) and a looplinking the two strands of the DNA double-stranded portion or stemcomprising or consisting of a linker selected from the group consistingof hexaethyleneglycol, tetradeoxythymidylate (T4) and2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane, the freeends of the DNA double-stranded portion or stem (i.e., at the oppositeof the loop) having three modified phosphodiester backbones (inparticular phosphorothioate internucleotidic links) and said Dbaitmolecule being conjugated to a cholesterol at its 5′ end, preferablythrough a linker (e.g., carboxamido oligoethylene glycol, preferablycarboxamido triethylene glycol).

The conjugated Dbait molecule or hairpin nucleic acid molecule can alsobe described by the following formulae:

wherein N is a nucleotide, n is an integer greater than 14, theunderlined N refers to a nucleotide having or not having a modifiedphosphodiester backbone, L′ is a linker, C is a molecule facilitatingendocytosis, L is a linker, and m is an integer being 0 or 1.Preferably, the underlined N refers to a nucleotide having a modifiedphosphodiester backbone. In Formulae (II) and (III), C-L_(m) isrespectively linked to the 5′ end or the 3′ end of the nucleotide. Informula (I-III), C-L_(m) is preferably linked to L′ through a disulfidebond (S—S).

In preferred embodiments, the molecule of formula (I), (II) or (III) hasone or several of the following features:

-   -   N is a deoxynucleotide, preferably selected from the group        consisting of A (adenine), C (cytosine), T (thymine) and G        (guanine) and selected so as to avoid occurrence of a CpG        dinucleotide and to have less than 80% or 70%, even less than        60% or 50% sequence identity to any gene in a human genome;        and/or    -   n is an integer from 15 to 195, preferably from 19-95, more        preferably from 21 to 95, and still more preferably from 27        to 95. In a particularly preferred embodiment, n is 27; and/or    -   the underlined N refers to a nucleotide having a        phosphorothioate or methylphosphonate backbone, more preferably        a phosphorothioate backbone; and/or    -   the linker L′ is selected from the group consisting of        hexaethyleneglycol, tetradeoxythymidylate (T4) and        2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane;        and/or    -   m is 1 and L is a carboxamido oligoethyleneglycol, more        preferably carboxamido triethylene glycol; and/or    -   C is selected from the group consisting of a cholesterol, single        or double chain fatty acids such as oleic acid or stearic acid,        ligands (including peptides, proteins, and aptamers), which        targets cell receptors such as folate, and transferrin,        preferably is a cholesterol, octadecyl, dioleoyl or folate, and        more preferably is a cholesterol.

Preferably, C-L_(m) is a triethyleneglycol linker(10-O-[1-propyl-3-N-carbamoylcholesteryl]-triethyleneglycol radical).

In a preferred embodiment, the conjugated Dbait molecule or hairpinnucleic acid molecule has the following formula:

with the same definition as formulae (I), (II) and (III) for N,underlined N, n, L, L′, C and m.

In a preferred embodiment, NNNN—(N)_(r)—N comprises at least 16, 18, 20,22, 24, 26, 28, 30 or 32 consecutive nucleotides of Dbait32 (SEQ ID NO:1), Dbait32Ha (SEQ ID NO: 2), Dbait32Hb (SEQ ID NO: 3), Dbait32Hc (SEQID NO: 4) or Dbait32Hd (SEQ ID NO: 5) or consists of 20, 22, 24, 26, 28,30 or 32 consecutive nucleotides of Dbait32 (SEQ ID NO: 1), Dbait32Ha(SEQ ID NO: 2), Dbait32Hb (SEQ ID NO: 3), Dbait32Hc (SEQ ID NO: 4) orDbait32Hd (SEQ ID NO: 5). In a particular embodiment, NNNN—(N)_(r)—Ncomprises or consists of Dbait32 (SEQ ID NO: 1), Dbait32Ha (SEQ ID NO:2), Dbait32Hb (SEQ ID NO: 3), Dbait32Hc (SEQ ID NO: 4) or Dbait32Hd (SEQID NO: 5), more preferably Dbait32Hc (SEQ ID NO: 4).

Accordingly, the conjugated Dbait molecule or hairpin nucleic acidmolecule may be selected from the group consisting of:

with the same definition as formulae (I), (II) and (III) for L, L′, Cand m.

In preferred embodiments, the molecule of formulae (Ia), (IIa), (IIIa),(Ib), (IIb), (IIIb), (Ic), (IIc), (IIIc), (Id), (IId), (IIId), (Ie),(IIe) and (IIIe), preferably formulae (II), (IIa), (IIb), (IIc), (IId)and (IIe), has one or several of the following features:

-   -   the underlined nucleotide refers to a nucleotide having or not        having a phosphorothioate or methylphosphonate backbone, more        preferably a nucleotide having a phosphorothioate or        methylphosphonate backbone, still more preferably a nucleotide        having a phosphorothioate backbone; and/or    -   the linker L′ is selected from the group consisting of        hexaethyleneglycol, tetradeoxythymidylate (T4) and        2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane;        and/or    -   m is 1 and L is a carboxamido oligoethylene glycol, more        preferably carboxamido triethylene glycol; and/or    -   C is selected from the group consisting of a cholesterol, single        or double chain fatty acids such as oleic acid or stearic acid,        ligands (including peptides, proteins, and aptamers) which        targets cell receptors such as folate, and transferrin, and        preferably is a cholesterol, octadecyl, dioleoyl or folate, more        preferably a cholesterol.

Preferably, C-L_(m) is a triethyleneglycol linker(10-O-[1-propyl-3-N-carbamoylcholesteryl]-triethyleneglycol radical).

In a specific embodiment of the Dbait molecules or hairpin nucleic acidmolecules of formulae (I), (II), (III), (Ia), (IIa), (IIIa), (Ib),(IIb), (IIIb), (Ic), (IIc), (IIIc), (Id), (IId), (IIId), (Ie), (IIe) and(IIIe), preferably formulae (II), (IIa), (IIb), (IIc), (IId) and (IIe),L′ is preferably selected from the group consisting ofhexaethyleneglycol, tetradeoxythymidylate (T4) and2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane.

In a specific embodiment of the Dbait molecules or hairpin nucleic acidmolecules of formulae (I), (II), (III), (Ia), (IIa), (IIIa), (Ib),(IIb), (IIIb), (Ic), (IIc), (IIIc), (Id), (IId), (IIId), (Ie), (IIe) and(IIIe), preferably formulae (II), (IIa), (IIb), (IIc), (IId) and (IIe),with C being cholesterol, C-L_(m) is the radical

In a preferred embodiment, the conjugated Dbait molecule or hairpinnucleic acid molecule is selected from the group consisting of (II),(IIa), (IIb), (IIc), (IId), and (IIe), wherein C-L_(m) is the radical

and wherein L′ is preferably selected from the group consisting ofhexaethyleneglycol, tetradeoxythymidylate (T4) and2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane, morepreferably2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane.

In a very specific embodiment, the Dbait molecule or hairpin nucleicacid molecule has the following formula:

wherein C-L_(m) is the radical

wherein L′ is2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane andwherein the underlined nucleotides have a phosphorothioate backbone.Accordingly, the molecule has the following structure and it is referredthereto in the Example section as “coDbait”.

One of the cholesterol-Dbait conjugates, named DT01, is a 64-ntoligodeoxyribonucleotide consisting of two 32-nt strands ofcomplementary sequences connected through a1,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane linker,with a cholesterol tetraethyleneglycol at the 5′ end and 3phosphorothioate internucleotide linkages at each of the 5′ and the 3′ends. In solution, the molecule forms an intra-molecular hairpin 32-bpdouble helix. This double-stranded (ds) DNA structure is essential forits biological activity, and is the active pharmaceutical ingredient(API). Molecular formula of the sodium salt: C₆₇₈H₈₂₀N₂₄₄Na₆₅O₃₉₂P₆₅S₆;molecular weight of the sodium salt: 22359.2 Da; molecular weight of thefree acid: 20931.4 Da. The molecule can also be represented as follows:

A very surprising aspect of the present invention for the molecule offormula (II), (IIa), (IIb), (IIc), (IId) or (IIe) is that, although theactivity of Dbait molecules needs the presence of at least one free end,the molecules facilitating endocytosis linked to the 5′ end do notdecrease the activity.

Accordingly, the present invention also relates to a conjugated Dbaitmolecule as disclosed above, a pharmaceutical composition comprising itand optionally a pharmaceutically acceptable carrier, a conjugated Dbaitmolecule as disclosed above for use in the treatment of cancer, alone orin combination with radiotherapy and/or chemotherapy of a DNA-damagingantitumoral agent, a method for treating cancer comprising administeringa therapeutically effective amount of a conjugated Dbait molecule asdisclosed above, and the use of a conjugated Dbait molecule as disclosedabove for preparing a medicament for treating cancer, as detailed below.

Endosomolytic Agent

Conjugated Dbait molecules or hairpin nucleic acid molecules arepreferably used here in combination with an endosomolytic agent (e.g.,chloroquine, fusogenic lipids, peptides, etc.). Indeed, treatment by anendosomolytic agent facilitates the release of conjugated Dbaitmolecules from endosomes. In addition, this particular combinationallows the obtention of further surprising effects, includingexceptional results with low toxicity in vivo as well as retarded andsustained Dbait-mediated activity.

In particular, the endosomolytic agents are capable of effecting thelysis of the endosome in response to a change in pH, and anencapsulating, or packaging, component capable of packaging atherapeutic agent to be delivered to cellular or subcellular components.Endosomolytic substances include, but are not limited to, quinolinecompounds, especially 4-aminoquinoline and 2-phenylquinoline compoundsand amino, thio, phenyl, alkyl, vinyl and halogen derivatives thereof,fusogenic lipids, peptides or proteins.

In a preferred embodiment, the endosomolytic agent is a small molecule.The basic endosomolytic agent may be selected from the group consistingof quinine, chloroquine, hydroxychloroquines, amodiaquins (camoquines),amopyroquines, primaquines, mefloquines, nivaquines, halofantrines,quinone imines and a combination thereof. Preferred endosomolytic agentsare quinoline endosomolytic agents including, but not limited to, thecompounds listed below with their chemical names:7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline (chloroquine);7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline(hydroxychloroquine);7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline;4-(4-diethylamino-1-methylbutylamino) quinoline;7-hydroxy-4-(4-diethyl-amino-1-methylbutylamino)quinoline;7-chloro-4-(4-diethylamino-1-butylamino)quinoline(desmethylchloroquine);7-fluoro-4-(4-diethylamino-1-butylamino)quinoline);4-(4-diethyl-amino-1-butyl amino)quinoline;7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-fluoro-4-(1-carboxy-4-diethyl-amino-1-butylamino)quinoline;4-(1-carboxy-4-diethylamino-1-butylamino) quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-fluoro-4-(1-carboxy-4-diethyl-amino-1-methylbutylamino)quinoline;4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;4-(4-ethyl-(2-hydroxy-ethyl)-amino-1-methylbutylamino)quinoline;7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; hydroxychloroquine phosphate;7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline(desmethylhydroxychloroquine);7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino) quinoline;7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;8-[(4-aminopentyl)amino-6-methoxydihydrochloride quinoline;1-acetyl-1,2,3,4-tetrahydroquinoline;8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride;1-butyryl-1,2,3,4-tetrahydroquinoline; 3-chloro-4-(4-hydroxy-alpha;alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline;4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxyquinoline;3-fluoro-4-(4-hydroxy-alpha; alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline;4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline;4-(4-hydroxy-alpha;alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline;4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline;3,4-dihydro-1-(2H)-quinolinecarboxyaldehyde; 1,1′-pentamethylenediquinoleinium diiodide; 8-quinolinol sulfate and amino, aldehyde,carboxylic, hydroxyl, halogen, keto, sulfhydryl and vinyl derivatives oranalogs thereof. Other agents are disclosed in Naisbitt et al. (1997, JPharmacol Exp Therapy 280:884-893) and in U.S. Pat. No. 5,736,557. In amore preferred embodiment, the endosomolytic agent may be selected fromthe group consisting of chloroquine, hydroxychloroquine,desmethylchloroquine, hydroxychloroquine phosphate, anddesmethyl-hydroxychloroquine, and is preferably chloroquine orhydroxychloroquine, more preferably chloroquine.

In another embodiment, the endosomolytic agent is a fusogenic lipid,peptide or protein. Indeed, numerous fusogenic lipids, peptides orproteins are known in the art. For instance, fusogenic lipids, peptidesor proteins are those disclosed in the following patent applications:WO10057160, US2007/0293449, US2006/0051405, WO10053489, and WO09126933.In particular, WO09/126933 provides fusogenic lipids, peptides andproteins at pages 23-29.

The inventors demonstrated the high antitumoral therapeutic efficiencyof the combination of a conjugated Dbait molecule with chloroquine,whereas they observed that the same amount of chloroquine alone or incombination with irradiation did not show any anti-tumoral activity.

Accordingly, the present invention concerns a pharmaceutical compositioncomprising a conjugated Dbait molecule or hairpin nucleic acid moleculeof the invention and an endosomolytic agent, more particularly for usein the treatment of cancer. The present invention also concerns aproduct comprising a conjugated Dbait molecule or hairpin nucleic acidmolecule of the invention and an endosomolytic agent as a combinedpreparation for simultaneous, separate or sequential use, moreparticularly for use in the treatment of cancer. Preferably, theendosomolytic agent is selected from the group consisting of chloroquineor hydroxychloroquine, and still more preferably is chloroquine.Preferably, the conjugated Dbait molecule or hairpin nucleic acidmolecule of the invention is any particular conjugated Dbait molecule asdescribed above. In one embodiment, the Dbait molecule or hairpinnucleic acid molecule of the invention is covalently linked to anendosomolytic agent, preferably chloroquine or hydroxychloroquine, andstill more preferably is chloroquine, in particular as disclosed inWO2007/040469. In another preferred embodiment, the endosomolytic agent,preferably chloroquine, is not conjugated (i.e., not covalently linked)to the conjugated Dbait molecule or hairpin nucleic acid molecule of theinvention.

The pharmaceutical compositions contemplated herein may include apharmaceutically acceptable carrier in addition to the activeingredient(s). The term “pharmaceutically acceptable carrier” is meantto encompass any carrier (e.g., support, substance, solvent, etc.) whichdoes not interfere with the effectiveness of the biological activity ofthe active ingredient(s) and is not toxic to the host to which it isadministered. For example, for parenteral administration, the activecompound(s) may be formulated in a unit dosage form for injection invehicles such as saline, dextrose solution, serum albumin and Ringer'ssolution.

The pharmaceutical compositions can be formulated as solutions inpharmaceutically compatible solvents or as emulsions, suspensions ordispersions in suitable pharmaceutical solvents or vehicles, or aspills, tablets or capsules that contain solid vehicles in a way known inthe art. Formulations of the present invention suitable for oraladministration may be in the form of discrete units such as capsules,sachets, tablets or lozenges, each containing a predetermined amount ofthe active ingredient; in the form of a powder or granules; in the formof a solution or a suspension in an aqueous or non-aqueous liquid; or inthe form of an oil-in-water or water-in-oil emulsion. Formulationssuitable for parenteral administration conveniently comprise a sterileoily or aqueous preparation of the active ingredient which is preferablyisotonic with the blood of the recipient. Every such formulation canalso contain other pharmaceutically compatible and nontoxic auxiliaryagents, such as stabilizers, antioxidants, binders, dyes, emulsifiers orflavouring substances. The formulations of the present inventioncomprise an active ingredient in association with a pharmaceuticallyacceptable carrier and optionally other therapeutic ingredients. Thecarrier must be “acceptable” in the sense of being compatible with theother ingredients of the formulations and not deleterious to therecipient thereof. The pharmaceutical compositions are advantageouslyapplied by injection or intravenous infusion of suitable sterilesolutions or by oral dosage by the digestive tract. Methods for the safeand effective administration of most of these chemotherapeutic agentsare known to those skilled in the art. In addition, their administrationis described in the standard literature.

The pharmaceutical composition of the invention is not a liposomalcomposition. In particular, the conjugated Dbait molecule of the productof the invention is not formulated in a liposomal composition.

In particular, the present invention also relates to a product, kit orcombined preparation comprising (a) one or more unit dosage forms of aDbait molecule or hairpin nucleic acid molecule as disclosed above, (b)one or more unit dosage forms of an endosomolytic agent as disclosedabove, and optionally (c) one or more unit dosage forms of aDNA-damaging anti-tumoral agent as disclosed below.

DNA Damaging Treatment

In addition to the conjugated Dbait molecules and the endosomolyticagent, the treatment may also further comprise an antitumoral treatment,preferably a treatment by a DNA-damaging agent or radiotherapy. TheDNA-damaging treatment can be radiotherapy or chemotherapy with aDNA-damaging antitumoral agent, or a combination thereof.

DNA strand breakage can be achieved by ionized radiation (radiotherapy).Radiotherapy includes, but is not limited to, γ-rays, X-rays, and/or thedirected delivery of radioisotopes to tumor cells. Other radiotherapiesinclude microwaves and UV irradiation. Other approaches to radiationtherapy are also contemplated in the present invention.

The DNA-damaging antitumoral agent is preferably selected from the groupconsisting of an inhibitor of topoisomerases I or II, a DNA crosslinker,a DNA alkylating agent, an anti-metabolic agent and inhibitors of themitotic spindles.

Inhibitors of topoisomerases I and/or II include, but are not limitedto, etoposide, topotecan, camptothecin, irinotecan, amsacrine,intoplicine, anthracyclines such as doxorubicine, epirubicine,daunorubicine, idanrubicine and mitoxantrone. Inhibitors oftopoisomerases I and II include, but are not limited to, intoplecin.

DNA crosslinkers include, but are not limited to, cisplatin, carboplatinand oxaliplatin.

Anti-metabolic agents block the enzymes responsible for nucleic acidsynthesis or become incorporated into DNA, which produces an incorrectgenetic code and leads to apoptosis. Non-exhaustive examples thereofinclude, without limitation, folic acid antagonists, pyrimidine analogs,purine analogs and adenosine deaminase inhibitors, and more particularlyMethotrexate, Floxuridine, Cytarabine, 6-Mercaptopurine, 6-Thioguanine,Fludarabine phosphate, Pentostatine, 5-Fluorouracil, Gemcitabine andCapecitabine.

The DNA-damaging anti-tumoral agent can be an alkylating agentincluding, without limitation, nitrogen mustards, ethyleniminederivatives, alkyl sulfonates, nitrosoureas, metal salts and triazenes.Non-exhaustive examples thereof include Uracil mustard, Chlormethine,Cyclophosphamide (CYTOXAN(R)), Ifosfamide, Melphalan, Chlorambucil,Pipobroman, Triethylenemelamine, Triethylenethiophosphoramine, Busulfan,Carmustine, Lomustine, Fotemustine, Cisplatin, Carboplatin, Oxaliplatin,Thiotepa, Streptozocin, Dacarbazine, and Temozolomide.

Inhibitors of the mitotic spindles include, but are not limited to,paclitaxel, docetaxel, vinorelbine, larotaxel (also called XRP9881;Sanofi-Aventis), XRP6258 (Sanofi-Aventis), BMS-184476(Bristol-Myers-Squibb), BMS-188797 (Bristol-Myers-Squibb), BMS-275183(Bristol-Myers-Squibb), ortataxel (also called IDN 5109, BAY 59-8862 orSB-T-101131; Bristol-Myers-Squibb), RPR 109881A (Bristol-Myers-Squibb),RPR 116258 (Bristol-Myers-Squibb), NBT-287 (TAPESTRY), PG-paclitaxel(also called CT-2103, PPX, paclitaxel poliglumex, paclitaxelpolyglutamate or XYOTAX), ABRAXANE (also called Nab-Paclitaxel; ABRAXISBIOSCIENCE), Tesetaxel (also called DJ-927), IDN 5390 (INDENA),Taxoprexin (also called docosahexanoic acid-paclitaxel; PROTARGA),DHA-paclitaxel (also called TAXOPREXIN), and MAC-321 (WYETH). Also seethe review of Hennenfent & Govindan (2006, Annals of Oncology, 17,735-749).

Cancers or Tumors to be Treated

The pharmaceutical compositions and the products, kits or combinedpreparations described in the invention can be used for treating cancerin a subject.

The terms “cancer”, “cancerous”, or “malignant” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, for example,leukemia, lymphoma, blastoma, carcinoma and sarcoma. More particularexamples of such cancers include chronic myeloid leukemia, acutelymphoblastic leukemia, Philadelphia chromosome-positive acutelymphoblastic leukemia (Ph+ALL), squamous cell carcinoma, small-celllung cancer, non-small cell lung cancer, glioma, gastrointestinalcancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer,endometrial cancer, kidney cancer, prostate cancer, thyroid cancer,neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervicalcancer, stomach cancer, bladder cancer, hepatoma, breast cancer, coloncarcinoma, head and neck cancer, gastric cancer, germ cell tumor,pediatric sarcoma, sinonasal natural killer, multiple myeloma, acutemyelogenous leukemia (AML), chronic lymphocytic leukemia, mastocytosisand any symptoms associated with mastocytosis.

“Leukemia” refers to progressive, malignant diseases of theblood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia is generally clinically classified onthe basis of (1) the duration and character of the disease—acute orchronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid(lymphogenous), or monocytic; and (3) the increase or non-increase inthe number of abnormal cells in the blood—leukemic or aleukemic(subleukemic). Leukemia includes, for example, acute nonlymphocyticleukemia, chronic lymphocytic leukemia, acute granulocytic leukemia,chronic granulocytic leukemia, acute promyelocytic leukemia, adultT-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylicleukemia, blast cell leukemia, bovine leukemia, chronic myelocyticleukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia,Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia,hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia,acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia,lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia,lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia,megakaryocyte leukemia, micromyeloblastic leukemia, monocytic leukemia,myeloblast leukemia, myelocytic leukemia, myeloid granulocytic leukemia,myelomonocytic leukemia, Naegeli's leukemia, plasma cell leukemia,plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia,Schilling's leukemia, stem cell leukemia, subleukemic leukemia, andundifferentiated cell leukemia. In certain aspects, the presentinvention provides treatment for chronic myeloid leukemia, acutelymphoblastic leukemia, and/or Philadelphia chromosome-positive acutelymphoblastic leukemia (Ph+ALL).

Various cancers are also encompassed by the scope of the invention,including, but not limited to, the following: carcinoma including thatof the bladder (including accelerated and metastatic bladder cancer),breast, colon (including colorectal cancer), kidney, liver, lung(including small and non-small cell lung cancer and lungadenocarcinoma), ovary, prostate, testes, genitourinary tract, lymphaticsystem, rectum, larynx, pancreas (including exocrine pancreaticcarcinoma), esophagus, stomach, gall bladder, cervix, thyroid, and skin(including squamous cell carcinoma); hematopoietic tumors of lymphoidlineage including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins'slymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, histiocyticlymphoma, and Burkitt's lymphoma; hematopoietic tumors of myeloidlineage including acute and chronic myelogenous leukemias,myelodysplastic syndrome, myeloid leukemia, and promyelocytic leukemia;tumors of the central and peripheral nervous system includingastrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin including fibrosarcoma, rhabdomyosarcoma, andosteosarcoma; other tumors including melanoma, xenoderma pigmentosum,keratoactanthoma, seminoma, thyroid follicular cancer, andteratocarcinoma; melanoma, unresectable stage III or IV malignantmelanoma, squamous cell carcinoma, small-cell lung cancer, non-smallcell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovariancancer, liver cancer, colorectal cancer, endometrial cancer, kidneycancer, prostate cancer, thyroid cancer, neuroblastoma, pancreaticcancer, glioblastoma multiforme, cervical cancer, stomach cancer,bladder cancer, hepatoma, breast cancer, colon carcinoma, head and neckcancer, retinoblastoma, gastric cancer, germ cell tumor, bone cancer,bone tumors, adult malignant fibrous histiocytoma of bone; childhoodmalignant fibrous histiocytoma of bone, sarcoma, pediatric sarcoma,sinonasal natural killer, neoplasm, plasma cell neoplasm,myelodysplastic syndromes, neuroblastoma, testicular germ cell tumor,intraocular melanoma, myelodysplastic syndromes;myelodysplastic/myeloproliferative diseases, and synovial sarcoma. Inaddition, disorders include urticaria pigmentosa; mastocytoses such asdiffuse cutaneous mastocytosis, solitary mastocytoma in human, as wellas dog mastocytoma, and some rare subtypes like bullous, erythrodermicand teleangiectatic mastocytosis; mastocytosis with an associatedhematological disorder, such as a myeloproliferative or myelodysplasticsyndrome, or acute leukemia; myeloproliferative disorder associated withmastocytosis; and mast cell leukemia, in addition to other cancers.Other cancers are also included within the scope of disorders including,but not limited to, the following: carcinoma, including that of thebladder, urothelial carcinoma, breast, colon, kidney, liver, lung,ovary, pancreas, stomach, cervix, thyroid, testis, particularlytesticular seminomas, and skin, including squamous cell carcinoma;gastrointestinal stromal tumors (“GIST”); hematopoietic tumors oflymphoid lineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin'slymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkitt'slymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; othertumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma andglioma; tumors of the central and peripheral nervous system, includingastrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, andosteosarcoma; and other tumors, including melanoma, xenodermapigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer,teratocarcinoma, chemotherapy refractory non-seminomatous germ-celltumors, and Kaposi's sarcoma, and any metastasis thereof.

In a preferred embodiment of the present invention, the cancer is asolid tumor. The term “solid tumor” especially means breast cancer,ovarian cancer, cancer of the colon and generally the GI(gastrointestinal) tract, cervical cancer, lung cancer, in particularsmall-cell lung cancer and non-small-cell lung cancer, head and neckcancer, bladder cancer, cancer of the prostate or Kaposi's sarcoma.

The pharmaceutical compositions and the products, kits or combinedpreparations described in the invention may be useful for inhibiting thegrowth of solid tumors, decreasing the tumor volume, and preventing themetastatic spread of tumors and the growth or development ofmicrometastases. The pharmaceutical compositions and the products, kitsor combined preparations described in the invention are in particularsuitable for the treatment of poor-prognosis patients or of radio- orchemo-resistant tumors.

The inventors tested a high number of different tumoral types for eachtumor (including melanomas, glioblastomas, and carcinomas) from celllines and patients' biopsies. More than 80% of them responded well tothe treatment. In particular, efficiency has been observed for thefollowing tumoral types: melanoma, glioblastoma, breast cancer, coloncancer, gastrointestinal cancer, liver cancer and head and neck cancer.

In a preferred embodiment, the cancer can be selected from melanoma,glioblastoma, breast cancer, colon cancer, gastrointestinal cancer,liver cancer and head and neck cancer.

Regimen, Dosages and Administration Routes

The effective dosage of each of the combination partners employed in thecombined preparation of the invention may vary depending on theparticular compound or pharmaceutical composition employed, the mode ofadministration, the condition being treated, and the severity of thecondition being treated. Thus, the dosage regimen of the combinedpreparation of the invention is selected in accordance with a variety offactors including the route of administration and the patient status. Aphysician, clinician or veterinarian of ordinary skill can readilydetermine and prescribe the effective amount of the single activeingredients required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentration of the activeingredients within the range that yields efficacy without toxicityrequires a regimen based on the kinetics of the active ingredients'availability to target sites.

The endosomolytic agent and the conjugated Dbait molecules may beadministered by the same route or by two distinct routes. Theadministration route for the endosomolytic agent, preferably chloroquineor hydroxychloroquine, more preferably chloroquine, and/or theconjugated Dbait molecules may be oral, parenteral, intravenous,intratumoral, subcutaneous, intracranial, intra-arterial, topical,rectal, transdermal, intradermal, nasal, intramuscular, intraperitoneal,intraosseous, or the like. In a preferred embodiment, the conjugatedDbait molecules are to be administered or injected near the tumoralsite(s) to be treated.

In a particular embodiment, the endosomolytic agent, preferablychloroquine or hydroxychloroquine, more preferably chloroquine, is to beadministered by an oral route or by an intraperitoneal route, preferablyby an oral route, and the conjugated Dbait molecules may be administeredby intratumoral injection, by subcutaneous injection, by intraperitonealinjection, by intracranial injection, by intravenous injection, or by anoral route, more preferably by intratumoral, subcutaneous or intravenousinjection, still most preferably by a subcutaneous route.

In another particular embodiment, the endosomolytic agent, preferablychloroquine or hydroxychloroquine, more preferably chloroquine, and theconjugated Dbait molecules are to be administered both by intratumoralinjection, by subcutaneous injection, by intraperitoneal injection, byintravenous injection, or by an oral route, more preferably byintratumoral, subcutaneous injection or by an oral route, still mostpreferably by an oral route. When the endosomolytic agent, preferablychloroquine or hydroxychloroquine, more preferably chloroquine, and theconjugated Dbait molecules are co-injected, the higher the amount of theendosomolytic agent, preferably chloroquine, the better the therapeuticeffect, within the limit of its toxicity. An advantage of theco-injection or local injection is that there is no need to match thepharmacokinetic profile in plasma. In a very specific embodiment, theendosomolytic agent, preferably chloroquine, is administered by an oralroute, and the conjugated Dbait molecules are to be administered bysubcutaneous injection. The inventors demonstrate exceptional resultswith this combination of administration routes, provided a pre-treatmentof chloroquine is administered in order to establish a steady-stateregimen of chloroquine in the plasma.

The endosomolytic agent, preferably chloroquine or hydroxychloroquine,more preferably chloroquine, is to be administered 2 hours before and/orsimultaneously with and/or after cholesterol-conjugated Dbait moleculesor hairpin nucleic acid molecules, more preferably 2 hours before theadministration of coDbait.

In a first preferred embodiment, the treatment regimen includes a stepof pre-treatment of the patient with the endosomolytic agent, preferablychloroquine, before the beginning of the treatment withcholesterol-conjugated Dbait molecules or hairpin nucleic acidmolecules. For instance, when the endosomolytic agent is administerednear the tumoral site to be treated (e.g., local administration), it canbe administered together or at least or about 1, 2, 3, 4 or 5 hoursbefore the administration of conjugated Dbait molecules or hairpinnucleic acid molecules, preferably between about one to three hoursbefore, more preferably about two hours before. Alternatively, when theendosomolytic agent is administered by systemic administration, it canbe administered longer before the administration of conjugated Dbaitmolecules or hairpin nucleic acid molecules and by a longer treatment,preferably during a period of about one to three weeks before theadministration of cholesterol-conjugated Dbait molecules or hairpinnucleic acid molecules, more preferably about a period of about twoweeks.

Once cholesterol-conjugated Dbait molecules or hairpin nucleic acidmolecules are or have been administered, the treatment with theendosomolytic agent can continue as long as the cholesterol-conjugatedDbait molecules or hairpin nucleic acid molecules are to beadministered. Alternatively, the treatment with the endosomolytic agentcan also end.

When a DNA-damaging antitumoral agent is used in combination with theconjugated Dbait molecule and the endosomolytic agent, the DNA-damagingantitumoral agent and the cholesterol-conjugated Dbait molecules may beadministered by the same route or by distinct routes. The administrationroute for the DNA-damaging antitumoral agent may be oral, parenteral,intravenous, intratumoral, subcutaneous, intracranial, intraarterial,topical, rectal, transdermal, intradermal, nasal, intramuscular,intraosseous, or the like.

In a particular embodiment, the DNA-damaging antitumoral agent and theendosomolytic agent are to be administered by an oral route,simultaneously, separately or sequentially, and the conjugated Dbaitmolecules may be administered by intratumoral injection, by subcutaneousinjection, by intraperitoneal injection, by intravenous injection, or byan oral route, preferably by intratumoral, subcutaneous orintraperitoneal injection or by an oral route, still more preferably byintratumoral or subcutaneous.

In another particular embodiment, the DNA-damaging antitumoral agent isto be administered by an oral route, and the conjugated Dbait moleculesand the endosomolytic agent may be administered, simultaneously,separately or sequentially, by intratumoral injection, by subcutaneousinjection, by intraperitoneal injection, by intravenous injection, or byan oral route, preferably by intratumoral, subcutaneous orintraperitoneal injection or by an oral route, still more preferably byintratumoral or subcutaneous.

In a further particular embodiment, the endosomolytic agent is to beadministered by an oral route, and the conjugated Dbait molecules andthe DNA-damaging antitumoral agent may be administered, simultaneously,separately or sequentially, by intratumoral injection, by subcutaneousinjection, by intraperitoneal injection, by intravenous injection, or byan oral route, preferably by intratumoral, subcutaneous orintraperitoneal injection or by an oral route, still more preferably byintratumoral or subcutaneous.

In an additional particular embodiment, the DNA-damaging antitumoralagent, the endosomolytic agent and the conjugated Dbait molecules are tobe administered, simultaneously, separately or sequentially, byintratumoral injection, by subcutaneous injection, by intraperitonealinjection, by intravenous injection, or by an oral route, preferably byintratumoral, subcutaneous or intraperitoneal injection or by an oralroute, still more preferably by intratumoral or subcutaneous injection.

The endosomolytic agent and conjugated Dbait molecules or hairpinnucleic acid molecules are to be administered before and/orsimultaneously with and/or after the irradiation and/or theadministration of the DNA-damaging antitumoral agent, more preferablybefore and/or simultaneously with the irradiation and/or theadministration of the DNA-damaging antitumoral agent. The irradiationand/or the administration of the DNA-damaging antitumoral agent isperformed so that the conjugated Dbait molecules are present in thetumoral cells when the irradiation is applied or when the DNA-damagingantitumoral agent reaches the tumoral cells. A physician, clinician orveterinarian of ordinary skill can determine the regimen based on theactive ingredients, their kinetics of availability to target sites ortheir pharmacokinetic profiles in plasma. Preliminary results indicatethat conjugated Dbait molecules stay active during one day. In a firstpreferred embodiment, the treatment regimen includes a step ofpre-treatment of the patient with the endosomolytic agent, preferablychloroquine or hydroxychloroquine, more preferably chloroquine, beforethe beginning of the treatment with the conjugated Dbait molecules orhairpin nucleic acid molecules. Then, the irradiation is to be appliedor the DNA-damaging antitumoral agent is to be administered at thebeginning of the treatment with the conjugated Dbait molecules orhairpin nucleic acid molecules or after the treatment with theconjugated Dbait molecules or hairpin nucleic acid molecules. Forinstance, the irradiation is to be applied or the DNA-damagingantitumoral agent is to be administered 3-24 h after the beginning ofthe treatment with the conjugated Dbait molecules. The DNA-damagingantitumoral agent and conjugated Dbait molecules may also besimultaneously administered.

Once the treatment by radiotherapy or with the DNA-damaging antitumoralagent has begun, the treatment with the endosomolytic agent and/orconjugated Dbait molecules can continue as long as the treatment byradiotherapy or with the DNA-damaging antitumoral agent is to be appliedor administered. Alternatively, the treatment with the endosomolyticagent and/or conjugated Dbait molecules can also end.

For conjugated Dbait molecules, the effective dosage of the DNA-damagingantitumoral agent employed in the combined preparation, kit or productof the invention may vary depending on the mode of administration, thecondition being treated, and the severity of the condition beingtreated. Thus, the dosage regimen of the conjugated Dbait molecules isselected in accordance with a variety of factors including the route ofadministration and the patient's status. A physician, clinician orveterinarian of ordinary skill can readily determine and prescribe theeffective amount of the conjugated Dbait molecules required to prevent,counter or arrest the progress of the cancer, in particular incombination with the selected DNA-damaging treatment.

For instance, for local administration (e.g., when intratumoral orsubcutaneous administration is used), the efficient amount of theconjugated Dbait molecules is at least 0.01 mg per 1 cm³ of tumor,preferably 0.1-40 mg per 1 cm³ of tumor, most preferably 1-20 mg per 1cm³ of tumor. The efficient amount can be administered in a dailytreatment protocol (e.g., 5 days per week for 3 to 6 consecutive weeksor 3 times a week for 3 to 6 consecutive weeks). Alternatively, anefficient amount of at least 0.1 mg per 1 cm³ of tumor, preferably0.1-40 mg per 1 cm³ of tumor, most preferably 1-20 mg per 1 cm³ oftumor, can be administered in a weekly treatment protocol for 3-6consecutive weeks, for instance. When other administration routes areused, one skilled in the art can adapt the amount in order to obtain anefficient amount of the conjugated Dbait molecules in the tumor of atleast 0.01 mg per 1 cm³ of tumor, preferably 0.1-40 mg per 1 cm³ oftumor, most preferably 1-20 mg per 1 cm³ of tumor, in particular in adaily treatment protocol or in a weekly treatment protocol. Forinstance, for a systemic route, the efficient amount or unit dosage ofthe conjugated Dbait molecules may be 0.1 to 100 mg, preferably 4 to 40mg. Accordingly, for a systemic route, the efficient amount or unitdosage of the conjugated Dbait molecules may be 0.06 to 0.6 mg/kg ofpatient. Of course, the dosage and the regimen can be adapted by oneskilled in art in consideration of the chemotherapy and/or radiotherapyregimen.

For the endosomolytic agent, in particular chloroquine orhydroxychloroquine, more preferably chloroquine, the effective dosage ofthe endosomolytic agent employed in the combined preparation, kit orproduct of the invention may vary depending on the mode ofadministration, the condition being treated, and the severity of thecondition being treated. Thus, the dosage regimen of the endosomolyticagent is selected in accordance with a variety of factors including theroute of administration and the patient's status. A physician, clinicianor veterinarian of ordinary skill can readily determine and prescribethe effective amount of the endosomolytic agent required to prevent,counter or arrest the progress of the cancer, in particular incombination with the conjugated Dbait molecules and the selectedDNA-damaging treatment.

In a particular embodiment, when an oral route is used and if theselected endosomolytic agent is known to be useful for treating orpreventing malaria, the endosomolytic agent, in particular chloroquineor hydroxychloroquine, more preferably chloroquine, is used with thesame dose and regimen as for treating or preventing malaria. Forinstance, if the selected endosomolytic agent is chloroquine orhydroxychloroquine, more preferably chloroquine, chloroquine orhydroxychloroquine may be administered at 100-600 mg per day, preferably200-400 mg per day, more preferably about 300 mg per day, once, twice,three times or four times a week. In a particular embodiment,chloroquine or hydroxychloroquine may be administered at about 100 mgper day during one or two weeks or at about 300 mg, twice a week duringone or two weeks.

In another particular embodiment, when a local route is contemplated,for instance a subcutaneous or intratumoral route, the endosomolyticagent, in particular chloroquine or hydroxychloroquine, more preferablychloroquine, may be used with 100-300 mg.

For radiotherapy, any radiotherapy regimen known in the art may be used,in particular stereotactic irradiation (e.g., 15 Gy) or fractionatedirradiation. The use of fractionated irradiation may be particularlyefficient; for instance irradiation may be applied every day or every2-5 days, preferably every 3-4 days, for a period of one, two, three,four, five or six weeks. The irradiation may be from 1 to 10 Gy,preferably from 2 to 5 Gy, in particular 2, 3, 4 or 5 Gy. For instance,fractionated irradiation of 15×2Gy for six weeks, or 4 to 6×5Gy for twoweeks may be contemplated. In a preferred embodiment, the contemplatedradiotherapy is a protocol with 4 irradiations of 5 Gy for two weeks.Different regimens or conditions of combined treatments of cancer withirradiation and Dbait molecules have been tested and allowed todemonstrate that the radio-sensibilization of tumors by Dbait moleculesdepends on the doses of Dbait molecules but not on the irradiationdoses.

For chemotherapy, the effective dosage of the DNA-damaging antitumoralagent employed in the combined preparation, kit or product of theinvention or in combination with the composition of the invention mayvary depending on the particular DNA-damaging antitumoral agentemployed, the mode of administration, the condition being treated, andthe severity of the condition being treated. Thus, the dosage regimen ofthe DNA-damaging antitumoral agent is selected in accordance with avariety of factors including the route of administration and thepatient's status. A physician, clinician or veterinarian of ordinaryskill can readily determine and prescribe the effective amount of theDNA-damaging antitumoral agent required to prevent, counter or arrestthe progress of the cancer.

The treatment may include one or several cycles, for instance two to tencycles, in particular two, three, four or five cycles. The cycles may becontinued or separated. For instance, each cycle is separated by aperiod of time of one to eight weeks, preferably three to four weeks.

Further aspects and advantages of the present invention will bedisclosed in the following experimental section, which should beregarded as illustrative and not limiting the scope of the presentapplication. A number of references are cited in the presentspecification; each of these cited references is incorporated herein byreference.

EXAMPLES

A Multiscale Comparison of Distribution and Activity of Short DNA(Dbait) Complexed with Polyethylenimine (PEI) or Bound to Cholesterol

Characterization of Dbait/Vector Complexes and Cell Uptake

It has been shown that PEI is able to form non-covalentinterpolyelectrolyte complexes with DNA, oligonucleotides and RNA. LongPEI chains are highly effective in gene transfection, but morecytotoxic. The inventors tested several PEI particle polyplexes withDbait and compared their activity to a modified Dbait covalently linkedto cholesterol (called coDbait). The coDbait was a Dbait moleculecovalently bound to a fatty chain of cholesterol that was used withoutadditional vectors. For each vector tested, the main goal of theinventors was to develop a formulation with the most homogeneousparticle size distribution at the highest Dbait concentration. Thediameter and surface charge of the particles were measured by dynamiclaser light scattering (DLS). Using a multimodal analysis, the inventorsfound that branched PEI (bPEI25K) with a mean size of 25 Kd and linearPEI with a size of 22 Kd (PEI22K) or 11 Kd (PEI11K) formed complexeswith Dbait with similar properties (Table 1).

TABLE 1 Fluorescence and cellular uptake of formulated Dbait ChloroquineDbait fluorescence^(a) × MCC^(b) cor MCC^(c) MCC^(b) cor MCC^(c)Molecules/Methods (100 mg/mL) (μg/mL) 10e−6/μg/mL 5 hrs 5 hrs 24 hrs 24hrs — − 0 2.78 ND 3.2 ND chloroquine + 0 3.12 Electroporation − 1.6 6.178.72 1.41 10.79 1.75 Electroporation + 16 6.17 8.6 1.39 ND ND Dbait −1.6 6 2.65 0.43 3.45 0.56 Dbait/PEI11K − 1.6 1.92 26.46 13.78 13.26 6.91Dbait/bPEI25K − 1.6 3.04 51.62 16.98 57.98 19.07 Dbait/PEI22K − 1.6 3.3254.49 16.41 42.74 12.87 Dbait/SuperFect − 1.6 6.41 220 34.32 203.7831.79 Dbait/SuperFect + CQ + 1.6 6.31 218 34.01 ND ND coDbait − 1.6 3.4710.02 2.89 ND ND coDbait + CQ + 1.6 3.47 20.71 5.97 ND ND coDbait − 163.47 65.74 18.95 64 18.44 coDbait + CQ + 16 3.47 236.43 68.14 214.361.76 coDbait − 32 3.47 145 41.79 ND ND coDbait + CQ + 32 3.47 390.38112.50 ND ND ^(a)fluorescence at FL2 value; ^(b)MCC = mean cellularcontent FL2 value (>3 experiments); ^(c)cor MCC = corrected cellularcontent FL2/fluorescence

Different ratios of PEI on Dbait were tested. The lowest ratio leadingto 100% Dbait complex was determined by gel shift assay. The N/P ratiosof 6, 6 and 9 were respectively chosen for PEI11K, PEI22K and bPEI25Kfor further studies. Dbait-PEI complex particles were stable over aperiod of one hour in sucrose 10%. The highly homogenous morphology ofspherical particles in the population (with sizes ranging from 125 to140 nm) was confirmed by transmission electron microscopy (FIG. 1A). Thepresence of salt in the dilution buffer at concentrations exceeding 0.8mg/mL, or prolonged storage, induced PEI complex aggregation. SuperFectcomplexes (60 μg SuperFect/μg Dbait) giving larger and polydisperseaggregates (>2 μm) were used as a positive control. The unchargedamphiphilic copolymer Lutrol did not form stable interacting complexeswith Dbait and was used as a negative control in some experiments.

The inventors monitored the cellular uptake of the different complexesusing a fluorescent cy3-modified Dbait. The initial fluorescence of thecy3-Dbait complex was monitored immediately before transfection. In PEIcomplexes, the Dbait fluorescence was 2 to 3-fold decreased, indicatingthat the compaction of the molecules with PEI might quench fluorescence(Table 1). coDbait was also less fluorescent than naked Dbait,indicating that cholesterol might interact with the cyanine on the samemolecule. SuperFect or Lutrol did not affect fluorescence. Cellularcontent of human transfected fibroblast cells was measured by flowcytometry analysis. Fluorescence distribution of cells treated withnaked Dbait or Dbait-Lutrol mixture was not different from untreatedcontrols, indicating that Dbait molecules did not enter spontaneouslyinto the cells. Electroporation was relatively inefficient andincreasing the concentration of Dbait did not improve the transfectionefficiency. All polycationic polymers (PEI and SuperFect) promotedefficient cellular uptake, but linear PEIs showed a wider distributionthan Dbait/SuperFect or Dbait/PEIb25K complexes. The coDbait enteredcells without the help of transfection factors but with a 10-fold lowerefficiency than Dbait/PEI. Increasing the 10-15 folds the concentrationof coDbait allowed efficient transfection (FIG. 1B).

One limitation of the DNA transfer efficiency is its retention inendosomes, which prevents it from interacting with its target or beingtranscribed. DNA must escape from normal endosomal pathways into thecell, which leads to degradation. Therefore, the efficiency of DNAdelivery is correlated not only with cellular uptake, but also withdestabilization and escape from endosomes. PEI is known to have a highbuffering capacity that facilitates DNA release from endosomes andlysosomes (“proton sponge hypothesis”). In contrast, coDbait requiresthe help of fusogenic agents such as chloroquine (CQ) to be efficientlyreleased from the endosomes. In order to improve transfectionefficiency, we added 100 μM CQ to the cells half an hour beforetransfection. CQ increased by 2-4 fold the cellular uptake of coDbait(FIG. 1). The amount of Dbait released into the cell was monitored bythe activation of the DNA-PKcs kinase triggered by its binding to theDbait molecules (Quanz et al., 2009, supra). Addition of cholesterol didnot affect the ability of Dbait to activate purified DNA-PK (FIG. 2A).In the cell, the DNA-PKcs kinase activation was monitored by the amountof H2AX phosphorylation, which has been shown to be strictly dependenton DNA-PK. Both Dbait/PEI and coDbait induced H2AX phosphorylation intreated cells (FIG. 2B). Branched and linear PEI/Dbait complexes rapidlypromoted H2AX phosphorylation (FIG. 2C) that reached a maximum 6 hoursafter the beginning of transfection and persisted 24 h aftertransfection. Dbait-induced kinase activity was very low afterelectroporation at any time (FIG. 2). High concentrations of coDbaitwere very inefficient to phosphorylated H2AX, and required at least 24hours to reach maximal value. Addition of CQ during transfectionincreased DNA-PKcs activation in coDbait-transfected cells up to thatobserved with 10-fold less Dbait/PEI (FIG. 2C). CQ did not increase theactivity in Dbait/PEI transfected cells, indicating that Dbait isefficiently released from endosomes when it is complexed to PEI. SincecoDbait cellular uptake did not increase between 5 hours and 24 hoursafter transfection, the slow activation of DNA-PK by coDbait reveals itsslow release from endosomes.

Cellular Uptake and Overall Toxicity in Zebrafish Early Embryos

Analyzing Dbait uptake and activity in cell cultures does not allowconclusions to be made about the drug diffusion, cellular uptake andactivity in the whole organism. The inventors assessed this issue byinjecting Dbait-cy3, either naked or with adjuvant, into theintercellular space of 1000-cell stage (stage 1K) zebrafish embryos(Kimmel et al., 1995, Dev Dyn 203:253-310). This protocol allowed the invivo observation by confocal microscopy of Dbait-cy3 distribution at thecellular and sub-cellular level as well as its activity on thefast-dividing cells of the early zebrafish embryo. Naked Dbait-cy3injected at the animal pole of stage 1K embryos rapidly diffusedthroughout the whole blastoderm and was no longer detected by 15 minutesafter injection. The addition of Lutrol allowed Dbait to be retained inthe extracellular space around the injection point but did notfacilitate cellular uptake. In the presence of SuperFect or PEI,numerous fluorescent patches were observed inside the cells, indicatingefficient cellular uptake. coDbait-cy3 showed another type of behaviorwith a strong and persistent staining of plasmic membranes together withpatchy intracellular fluorescence. Embryos incubated with CQ prior toinjection converted the large coDbait fluorescent patches into diffuseintracellular distribution.

The observation of phenotypic effects 20 hours after injection allowedassessment of Dbait activity and treatment overall toxicity. Dbaitfluorescence was detected 24 hours after injection in the head cells oflarvae (FIG. 3A-C), which according to the zebrafish fate mapdevelopment (Woo et al., 1995, Curr Opin Genet Dev 5:439-443) derivedfrom the injected area at the animal pole of pre-gastrulation embryos.Injection of Dbait without adjuvant (NA) or combined with Lutrol (Lu)showed no effect on development (FIG. 3D), correlating with the poorintracellular uptake of Dbait described above. Addition of adjuvant ledto cell death in the head and, correlating with the injected volume,extensive cell death and teratogenesis might be observed. The 24-hourphenotypes were categorized as described (FIG. 3), allowingquantification of the injected mixture's toxicity. For the sameconcentration of Dbait, clear differences appeared in terms of celldeath and subsequent developmental abnormalities, depending on theadjuvant. Addition of SuperFect (sup) was very toxic to embryonic cellsand extensive cell death at early stages resulted in a high percentageof type 2 phenotypes. Similarly, addition of PEI (25K, 22K, 11K) provedto be toxic to zebrafish blastomeres. Although less efficient than PEIaddition, coDbait injection resulted in significant cell death. Embryopreincubation with CQ did not increase significantly the toxicity.Altogether, early embryonic cell death and subsequent developmentalabnormalities was a fast and reliable protocol to assessDbait+/−adjuvant's overall toxicity in zebrafish embryos. Correlation ofcell death with cellular uptake suggested that anti-tumoral activity ofDbait in embryonic cells might play an important role in the toxiceffect.

Local and Systemic Toxicity in Mice

To assess the consistency of cell culture, zebrafish embryos and mousedata, the inventors analyzed the tolerance of nude mice's skin torepeated administration of Dbait/PEI11K, Dbait/PEI22K, Dbait/bPEI25K,and coDbait. The toxicity of the different formulations of Dbait wasanalyzed after 3 daily subcutaneous (SC) injections. All the Dbait/PEIshowed high toxicity with injections that were tolerated at 3.75 mg/kgbut started at 5 mg/kg to trigger local inflammation associated withlocal necrosis and ischemia that rapidly disappeared with the arrest ofthe treatment. Intravenous (IV) injection toxicity gave similar results:the Dbait/PEI intravenous injections were lethal at 3 mg/kg with deathoccurring during injection, probably by blood clogging. Slow injectionsby perfusion (0.4 μL/mn) increased tolerance up to 6 mg/kg Dbait/PEI (6nmoles/injection), confirming that most of the IV toxicity is due tolocal concentration at the bolus injection site. coDbait with or withoutCQ did not show any toxicity at all tested doses (up to 800mg/kg/injection; 800 nmoles/injection) whatever the route used: SC, IVbolus or IV perfusion.

Antitumoral Activity in Xenografted Tumours

The antitumoral effect of formulated Dbait was tested in combinationwith radiotherapy on SK28 xenografted human melanoma. Dbait/vectorcomplexes were administered 5 hours before each irradiation usingintratumoral injections.

Though drug administration by intratumoral injection (IT) has been usedin many trials, it is currently advised to avoid that route of deliveryin clinical assays. The inventors investigated how Dbait/PEI11k orcoDbait could be administered by subcutaneous injection (SC) in the areanext to the tumor. Several clinical assays have successfully used thisroute of administration. The inventors first compared the diffusion ofthe molecules in tumors treated by one intratumoral injection or twosubcutaneous injections performed at opposite sides of the tumor (FIG.4). Fluorescent Dbait complexed to PEI11k tended to form aggregates atthe site of injection and diffused progressively to the edge of thetumor. In contrast, coDbait showed a more even distribution around theinjection, whether inside the tumor or in its vicinity. SC injections ofDbait/PEI11k or coDbait were slightly less efficient than IT injectionsin terms of tumor growth control (Table 2). However, increasing thenumber of injection sites should allow significant improvement of tumorgrowth control without adding local toxicity.

TABLE 2 Xenografted mice's survival after irradiation associated withvarious treatments Median Dbait/Vector Dbait mode of Nb. Cured survivalRelative risk Mean SD Mean % complexes concentration admin.^(a) micemice^(b) time (p value) TGD TGD TGD^(c) Mock — IT 79 2 72 — 11 15 160Dbait 6 × 60 μg (3 nmol) IT 6 0 63 1.40 (p < 0.41) 3 8 114 Dbait/PEI11k6 × 60 μg (3 nmol) IT 38 3 123 0.26 (p < 2.69.10−8) >40 27 313Dbait/PEI22k 6 × 60 μg (3 nmol) IT 10 2 >150 0.09 (p < 4.62.10−6) >63 40436 Dbait/PEIb25k 6 × 60 μg (3 nmol) IT 19 4 >150 0.13 (p <3.73.10−8) >60 35 419 Dbait/Lutrol 6 × 60 μg (3 nmol) IT 10 0 72 1.2 (p< 0.56) 5 5 128 Dbait/PEI11k 6 × 150 μg SC 12 0 135 0.34 (p < 1.05.10−3)22 15 216 (7.5 nmol) CQ — IT 6 0 68 — 8 6 142 coDbait + CQ 6 × 600 μg IT13 0 98 0.23 (p < 4.02.10−4) 42 25 323 (30 nmol) coDbait + CQ 6 × 1.2 mgSC 16 0 101 0.22 (p < 1.05.10−3) >22 23 218 (60 nmol) ^(a)mode ofadministration: IT, intratumoral; SC, subcutaneous ^(b)cured mice areanimals with no recurrence within 300 days following treatment ^(c)TGDcalculation and statistical analysis are described in Material andMethods

Survival of five groups of nude mice bearing SK28 melanoma xenograftshas been studied. Group 1) untreated mice (n=16); Group 2) irradiatedmice (IR, n=12); Group 3) irradiated mice with intraperitoneallyinjected 1 mg chloroquine (CQ, IR, n=10); Group 4) treated mice byintratumorally injected 0.6 mg DT01 (also called coDbait) and irradiated5 hours later (DT01, IR, n=11); and Group 5) pretreated mice withintraperitoneally injected 1 mg chloroquine 2 hours prior tointratumoral injection of 0.6 mg DT01 (also called voDbait) andirradiated 5 hours later (DT01, CQ, IR, n=13).

Results are presented in FIG. 5.

With 0.6 mg coDbait administered intratumorally, pre-treatment bychloroquine remarkably radiosensitized and increased the survival (group5) as compared to radiotherapy alone (group 2), while neither coDbait(group 4) nor CQ (group 3) showed significant radiosensitization. Theextent of radiosensitization of group 5 was similar to that treated by0.06 mg Dbait formulated with polyethylenimine (PEI) at the ratio N/P=6.

An administration regimen based on a subcutaneous injection of coDbaithas also been evaluated on nude mice bearing SK28 melanoma xenografts.This regimen is schematically disclosed in FIG. 6. In brief, thisregimen includes four combined treatments by coDbait and irradiation intwo weeks. In particular, 4 mg of coDbait were subcutaneously injectedat two opposite points separated by 5 mm from the tumor border. Inaddition, the animals have been pretreated with 1 mg of chloroquine (CQ)and further treated with CQ at the same dosage during the treatment withcoDbait and irradiation. Tumor growth was evaluated after thisadministration regimen and the results are given in FIG. 6.

It has been observed that the lowest tumor growth is observed with thecotreatment of coDbait and irradiation with chloroquine after apretreatment with chloroquine. In addition, the group co-treated withchloroquine showed more homogenous results than those withoutchloroquine treatment.

Conclusion

In the present study, the inventors used a set of assays to guidedevelopment of administration protocols and drug formulation. Theseassays allowed the comparison of different formulations of Dbait beforeperforming preclinical assays on mice. Cellular and zebrafish embryoassays were used to assess the efficiency of Dbait cellular uptake, aprerequisite step in the antitumoral drug effect, and to select the mostappropriate protocols and formulations for preclinical studies onmammals. Overall toxicity in zebrafish embryos did not correlate withtoxicity in mice's skin or after systemic injection. In particular, thehigh toxicity of coDbait in zebrafish embryos indicated that most of thecells contacting the drug probably died, whereas mice's skin did notshow any reaction to injections of high doses of coDbait. Thisdifference suggests that toxicity in zebrafish early embryos is anindicator of tumor sensitivity rather than healthy tissue sensitivity.Actually, Dbait molecules have been shown to be specifically toxic intumors but not in normal skin (Quanz et al., 2009, supra). Dbait/PEI (5μM) and coDbait (50 μM)+CQ that triggered comparable DNA-PKcs activationin cell cultures had a similar toxic effect on zebrafish embryos (FIG.3D) and displayed significant antitumoral activity on mouse tumors(Table 2, FIGS. 5 and 6). This observation is consistent with thesensitivity to antitumoral activity of zebrafish embryonic cells thatshare characteristic properties with tumor cells, including mitoticindex and biochemical and phenotypic traits. In agreement with thishypothesis, the inventors recently demonstrated Dbait antiproliferatingactivity by direct intracellular injection of naked Dbait into zebrafishblastomeres between the 1 and 16-cell stage.

PEI polymers were among all tested adjuvant molecules, the mostefficient in forming Dbait complexes. Their use was however limited bytheir toxicity on tissues as well as in the blood system. Local toxicitywas partly overcome by slow administration (perfusion) and by splittingthe injected doses between different injection sites. But the covalentcombination of cholesterol and Dbait provided the best alternative forDbait delivery to the cells without adjuvant addition. Indeed, the lackof toxicity within the range of tested doses suggests that this moleculemight prove to be useful despite the highest doses required forantitumoral effect. The doses of 3 nmoles and 30 nmoles per injection ofDbait/PEI11K and coDbait respectively doubled the delay in tumour growthinduced by irradiation alone. The respective toxicity of bothformulations (6 nmoles and >800 nmoles) gave a relative ratio ofefficiency dose/toxicity dose of 0.5 for Dbait/PEI11K and <0.037 forcoDbait, indicating that coDbait is a very good candidate for clinicaltrials.

Materials and Methods

Dbait and Particle Formation

Dbait and coDbait molecules were obtained by automated solid-phaseoligonucleotide synthesis from Eurogentec (Seraing, Belgium) or fromAgilent Technologies Nucleic Acid Solution Division (Boulder, USA) asdescribed previously (Quanz et al., 2009, supra). They were purified bydenaturing reverse-phase HPLC and/or HPLC-IEX. Some Dbait derivativeswere labeled with the fluorophores Cy3 (λ_(excitation)=540 nm;λ_(emission)=560 nm) or Cy5.5 (λ_(excitation)=X nm; λ_(emission)=X nm).Linear PEI (11 kDa and 22 kDa) were from Polyplus-transfection(Illkirch, France) and provided as a ready-to-use solution of 300 mMnitrogen concentration. Branched bPEI25 kd was purchased fromSigma-Aldrich (Saint Quentin, France). Lutrol was purchased fromIn-Cell-Art (Nantes, France). Dbait and PEI solutions (stock PEI) werediluted in 10% sucrose or 150 mM NaCl (for in vitro transfectionexperiments) to obtain various ratios of vector/Dbait. The ratio ofPEI/Dbait (or ratio N/P) was determined according to the number of aminenitrogen for PEI and phosphate for Dbait. Typically, for 300 μL ofcomplexes at 0.6 mg/mL and N/P 6, Dbait (180 μg, 0.54 μmol of phosphate)and the desired amount of polymer solution (11.4 μL of PEI stocksolution contains 0.3 μmol of amine nitrogen) were each diluted into 150μL (10% sucrose). SuperFect/Dbait particles were prepared according tothe manufacturer (Qiagen, Courtaboeuf, France) in a ratio of 10 μlSuperFect per μg DNA. The complexation of vector/Dbait was analyzed bythe agarose gel electrophoresis method. The samples (18 μL) were mixedwith bromophenol blue dye (1 μL) and then loaded on 1.5% agarose gelinto the electrophoresis chamber containing TAE buffer 1× (40 mMTris-acetate, pH 8.3, 1 mM EDTA). The gel was run at 100 volts for 30min. Then the gel was stained with ethidium bromide (EtBr) for 15minutes and the bands observed under UV light.

Cell Culture, Dbait Molecules and Transfection

The Dbait molecules were made by automated solid-phase oligonucleotidesynthesis. Sequence is5′-GCTGTGCCCACAACCCAGCAAACAAGCCTAGA-(H)-TCTAGGCTTGTTTGCTGGGTTGTGGGCACAGC (SEQ ID NO: 4) where H is a hexaethyleneglycollinker. Studies on cells in culture were performed using SV40transformed fibroblasts MRC-5. Cells were grown at 37° C. in monolayercultures in complete DMEM (Gibco, Cergy-Pontoise, France) with 10% FCSand antibiotics (100 μg/mL streptomycin and 100 μg/mL penicillin) underconditions of 100% humidity, 95% air and 5% CO₂. Unless otherwisespecified, transfections were performed in 1.2 mL MEM medium withoutserum in 60 mm diameter plates. Transfection with jetPEI(Polyplus-transfection, Illkirch, France) was performed at an N/P ratioof 6 according to the manufacturer's instructions. Briefly, Dbait wasdiluted in 150 mM NaCl and gently mixed with an equal volume of PEI in150 mM NaCl and added to DMEM medium without serum. coDbait was directlyadded to DMEM medium without serum. Transfection of Dbait molecules wasperformed with SuperFect reagent in 1.2 mL DMEM medium with serum (in 60mm diameter plates) for 5 hours and then cells were left to recover for1 hour if not indicated otherwise. For electroporation, 1.2×10⁶ cellswere transfected with 2 μg Dbait using the Gene Pulser II (Bio-Rad,Marnes-la-Coquette, France). At the end of the 5 h transfection (timezero), medium was replaced by complete medium and cells were grown forthe indicated time before being analyzed. Chloroquine (50 μM) was added30 min before transfection.

Flow Cytometry

Cells were transfected with different complexes with Dbait-cy3 for 5 hand allowed to grow 5 hours or 24 hours, then rapidly washed with PBS.Cells were directly analyzed by flow cytometry. For immunofluorescencedetection by flow cytometry, the cells were fixed in 2% paraformaldehydefor 10 min prior to immunodetection. Note that permeabilizationtreatment removed most of the Dbait, impairing immunofluorescencedetection and Dbait detection on the same cells. Cells were fixed for 15min in 4% formaldehyde, permeabilized in 0.2% Triton X-100 for 1 hour,blocked with 2% BSA and incubated with primary antibody for 2 hours onice with mouse monoclonal antibodies anti γ-H2AX (Upstate Biotechnology,Temecula, Calif., USA) and revealed with secondary antibodies conjugatedwith Alexa-488 (Molecular Probes, Eugene, Oreg., USA) and Texas Red(Rockland, Gilbertsville, Pa., USA) at a dilution of 1/200 for 30 min atRT. Cells were washed with PBS and resuspended in PBS with 50 μg/mLpropidium iodide, 25 μU/ml RNaseA. Cells were analyzed by a FACSCaliburflow cytometer (BD Biosciences, Franklin Lakes, N.J., USA) and data wasanalyzed using BD CellQuest Pro (BD Biosciences) and the free WinMDI 2.8(Scripps Research Institute, La Jolla, Calif., USA) software.

Zebrafish Husbandry, Embryo Collection and Treatment

Zebrafish eggs were obtained from natural spawning of wild type ortransgenic (βactin:egfp-ras) fish lines. Narishige (MN 153)micromanipulator fixed on a dissecting scope with epifluorescenceillumination (Leica MZ16F) and air injector (Eppendorf FemtoJet) wereused to perform Dbait injection at cell stage 1K. Glass capillaries(Harvard Apparatus GC100-10) were pulled with a KopF vertical pipettepuller (KopF 720) to make injection needles. 2 to 5 nl of Dbait solutionwere injected at the animal pole of embryos in cell cycle 10 andimmediately processed for confocal laser scanning microscopy imaging(upright Leica SP2) with 40×/0.8 NA water dipping lens objective.Imaging was performed by simultaneous 480 nm (eGFP) and 561 nm (cy3)excitation. Embryos were further grown at 28.5° C. until 24 hpf.One-day-old larvae were observed under the dissecting stop andphenotypes categorized as described in FIG. 3. Chloroquine treatmentprior to injection consisted of 2 hours of incubation in embryo medium(The Zebrafish Book) with 50 chloroquine. Dbait (coDbait)-cy3 50 μM wasinjected either alone or in combination with PEI25K (ratio N/P=9),PEI11K (ratio N/P=6), or SuperFect (10 μg Dbait).

Dbait and Irradiation Treatments in Mice

SK28 or U87G xenograft tumors were obtained by injecting 10⁶ tumor cellsinto the flanks of adult female nude mice (Charles River strain;L'Arbresle, France). The animals were housed in the laboratory at leastone week prior to commencing experiments. There were 5-6 animals percage under controlled conditions of light and dark cycles (12 hrs:12hrs), relative humidity (55%) and temperature (21° C.). Food and tapwater were available ad libitum. After approximately 12 days, when thesubcutaneous tumors measured 150-200 mm³, the mice were separated intohomogeneous groups of at most 12 each to receive different treatments.Irradiation was performed in a ¹³⁷Cs unit (0.5 Gy/min) with a shielddesigned to protect about two-thirds of the animal's body. Doses werecontrolled by thermoluminescence dosimetry. A total dose of 30 Gy wasdelivered in 6 sessions at intervals of three sessions of 5 Gy per weekduring two weeks. Dbait molecules were prepared in 100 μL of 10% sucroseas previously described for in vitro studies except that PEI mixtureswere performed without NaCl (Polyplus-transfection, Strasbourg, France).The Dbait mixtures were incubated for 15 min at room temperature beforeinjection. CoDbait was diluted in 10% sucrose at the requiredconcentration. Intratumoral injections of the indicated amount of Dbaitwere performed 5 h before each radiotherapy session. Mock treatedanimals were injected with 100 μL of 10% glucose according to theprotocol of the associated assays. Tumor size was assessed by calipermeasurements every three days and size was calculated by the formula(2×length×width²). Mice were weighed and pictures of tumors were takenevery week. For ethical reasons, the animals were sacrificed when theirtumors reached 2,000 mm³. The end point used in survival analysis wasdeath day. The Local Committee on Ethics of Animal Experimentation(Orsay, France) approved all experiments.

Statistical Analysis

Descriptive analyses of the tumor response were performed for eachtreatment and each tumor type. Day 1 was the day of the first treatmentsession. All animals were followed for at least 150 days. Medianlifetime was estimated according to the Kaplan-Meier method. Tumorgrowth delay (TGD) was calculated by subtracting the mean tumor volumequadrupling time of the control group from tumor volume quadruplingtimes of individual mice in each treated group. The mean TGD wascalculated for each treated group using the individual measurements.Overall survival curves were assessed by Kaplan-Meier estimates andcompared using the non-parametric LogRank test since the data do notfollow a normal distribution. The analysis was performed using statELsoftware (ad Science, Paris, France). A global LogRank was firstperformed for each group with the same tumor type. Then treatments withDbait were compared to the mock-treated controls. The number of animals(n), the relative risk (RR) and the p value are reported in Table 2. Alltests were considered significant at the 0.05 significance level.

Physico-Chemical Properties of Formulated Dbait Particles

The particle size of vector/Dbait was determined by dynamic lightscattering (DLS) on the Zetasizer Nano Series (Malvern Instruments,Paris, France) with these specifications: medium viscosity: 1.150 cP,refractive index: 1.45, scattering angle: 90°, temperature: 25° C. Dataare the mean of 3-5 measures per sample with each measure averaging thedata of 10-15 sub-runs. Data were analyzed using the multimodal numberdistribution software supplied with the instrument. For thezeta-potential measurement, particles were diluted in 10% sucrose/10 mMNaCl to give a final Dbait concentration of 0.1 mg/mL and were measuredwith the following specifications: 3 measurements, medium viscosity:1.054 cP, medium dielectric constant: 79, temperature: 25° C.

Cationic Ratio^(a) Zeta^(c) [C]_(max) polymers (w/w) Size^(b) (nm) (mV)Pdl (mg/mL) supplier Superfect — >1000 — 1 nd Qiagen bPEI25K 2.1 175 ±51 +40 0.11 1.5 Sigma- Aldrich PEI22K 1.4 133 ± 25 +46 0.17 1.0Polyplus- transfection PEI11k 1.4 125 ± 13 +30 <0.2 0.8 Polyplus-transfection ^(a)weight ratio at which maximum Dbait activity isobserved ^(b)Mean diameter (+/− SD) as determined by dynamic lightscattering (see supplementary Material and Methods). ^(c)Particles insucrose 1%, 10 mM NaCl

Transmission Electron Microscopy

Samples were prepared for transmission electron microscopy by negativestaining with uranyl acetate. A drop of sample (10 μL) was deposited onthe grid (formvar/carbon on 200 mesh copper, Agar Scientific) and leftfor 3 minutes before removing excess liquid with blotting paper. Thenthe complexes were stained with 10 μL of aqueous uranyl acetate (2%) for2 min and the excess was removed with blotting paper. Observations wereperformed with a Jeol JEM-100S Electron Microscope.

Alternative Conjugated Dbait Molecules

Alternative conjugated Dbait molecules have been prepared and aredescribed as follows:

Conjugated molecules of formula (IIe)

with

Measured mass (MALDI- Compound C-Lm L′ TOF) 0902Cholesterol-triethyleneglycol hexaethyleneglycol 20830.5 09031,2-di-(9Z-octadecenoyl)-sn-glycero-3- hexaethyleneglycol 21306.9phosphoethanolamine-N-[4-(p-phenyl-3- ((6-phosphohexylthio)-succinimido))butyramide] 0904 N-octadecyl-hexylthioamidehexaethyleneglycol 20411.6 0905 N-hexyl-folic acid-amidehexaethyleneglycol 20739.2

Conjugated molecules of formula (Ie)

with

Measured mass Compound C-Lm L′ (MALDI-TOF) 0813 Cholesterol-N-(5-hydroxymethyl- 21127.7 triethyleneglycol 6-phosphohexyl)-11- (3-(6-phosphohexythio) succinimido)) undecamide 0815 Cholesterol- 1,3-bis-[5-21454.0 dihexylsulfide hydroxylpentylamido] propyl-2-(6- phosphohexyl)

The activity of these alternative conjugated Dbait molecules has beenmeasured through DNA PK inhibition as detailed above (FIG. 7). It hasbeen observed that the conjugated molecules maintain their activity. Inparticular, the conjugation of various lipids and ligands at either the5′ end or in the loop has minor impact on the capacity of thesemolecules to trigger DNA-PK activity.

In addition, their activity has also been measured on cell lines, withor without chloroquine, through the determination of the amount of H2AXphosphorylation as detailed above (FIG. 8). First, for the testedconjugated Dbait molecules, it has been observed that their activity ishigher with a prior treatment with chloroquine. In addition, it can benoted that the conjugation of the cholesterol to the 5′ end surprisinglyleads to more effective molecules than the conjugation of thecholesterol in the hairpin loop (see 0902 in comparison to 0813 and0815).

Cellular Uptake of Conjugated Dbait Molecules

Cellular uptake of the Dbait conjugated to cholesterol, in particularCoDbait, in comparison with the Dbait was measured by the inventors byflow cytometry analysis.

The results are given in the following table.

Cy3 fluorescence intensity @5 hrs. Transfection conditions Median MeanDbait-Cy3 79 nM 3 3 Dbait-Cy3 79 nM + Superfect 426 327 CoDbait-Cy3 79nM 10 10 CoDbait-Cy3 79 nM + CQ 50 μM 20 21 CoDbait-Cy3 986 nM 63 66CoDbait-Cy3 986 nM + CQ 50 μM 239 236 Co_siRNA_H2AX-Cy3 79 nM 53 56Co_siRNA_H2AX-Cy3 79 nM + CQ 50 μM 60 63 Co_siRNA_H2AX 986 nM 661 710Co_siRNA_H2AX-Cy3 986 nM + CQ 50 μM 1144 1214

Flow cytometry analysis of cellular uptake in the MRC5 cell lineperformed at 5 hours after the beginning of treatment of varioustransfection conditions was as described in the table. Alloligonucleotides were labeled by cyanine 3 (Cy3) dye: Dbait (Dbait-Cy3),cholesterol-Dbait (0813)(CoDbait-Cy3) and siRNA targeting H2AX with acyanine 3 and a cholesterol at the 5′ and 3′ of the sense strand(Co_siRNA_H2AX: Cy3-5′-CAACAAGAAGACGCGAAUCTT-3′-cholesterol (SEQ ID NO:6); 5′-GAUUCGCGUCUUCUUGUUGTT-3′ (SEQ ID NO: 7). When indicated, 50 μM ofchloroquine (CQ) was added prior to transfection.

We claim:
 1. A conjugated nucleic acid molecule which has the followingformula:

wherein N is a nucleotide, n is an integer from 15 to 195, theunderlined N refers to a nucleotide having or not a modifiedphosphodiester backbone, L′ is a linker; m is 1 and L is a linker, C isa molecule facilitating endocytosis selected from the group consistingof single or double chain fatty acids, tocopherol, folates or folicacid, cholesterol, sugar, peptide and protein, and wherein C isconjugated via L to the 5′ end of the nucleic acid molecule.
 2. Theconjugated nucleic acid molecule according to claim 1, wherein n is 27.3. The conjugated nucleic acid molecule according to claim 1, wherein Cis selected from the group consisting of tocopherol and cholesterol. 4.The conjugated nucleic acid molecule according to claim 1, wherein L′ isa loop linking the two strands of the nucleic acid molecule.
 5. Theconjugated nucleic acid molecule according to claim 1, wherein L′ isselected from the group consisting of a nucleotide linker and anon-nucleotide linker.
 6. The conjugated nucleic acid molecule accordingto claim 5, wherein L′ is a non-nucleotide linker selected from thegroup consisting of abasic nucleotide, polyether, polyamine, polyamide,peptide, carbohydrate, lipid, polyhydrocarbon and polymeric compounds.7. The conjugated nucleic acid molecule according to claim 5, wherein L′is selected from the group consisting of hexaethyleneglycol,tetradeoxythymidylate (T4),1,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane and2,19-bis(phosphor)-8-hydraza-1-hydroxy-4-oxa-9-oxo-nonadecane.
 8. Theconjugated nucleic acid molecule according to claim 1, wherein L isselected from the group consisting of aliphatic chain, polyether,polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon,polymeric compounds and compounds incorporating a disulfide linkage, aprotected disulfide linkage, an amide linkage, an acid labile linkage,an ester linkage, an ortho ester linkage, a phosphonamide linkage, abiocleavable peptide linkage, an azo linkage and an aldehyde linkage. 9.The conjugated nucleic acid molecule according to claim 8, wherein L isselected from the group consisting of an oligoethylene glycol and adialkyl-disulfide.
 10. The conjugated nucleic acid molecule according toclaim 1, wherein the molecule is

with 3 phosphorothioate internucleotide linkages at each of the 5′ andthe 3′ ends.
 11. A pharmaceutical composition comprising a conjugatednucleic acid molecule according to claim
 1. 12. The pharmaceuticalcomposition according to claim 11, wherein said composition furthercomprises a DNA-damaging anti-tumoral agent and a pharmaceuticallyacceptable carrier.
 13. A kit comprising a conjugated nucleic acidmolecule according to claim 1 and a DNA-damaging anti-tumoral agent, asa combined preparation for simultaneous, separate or sequential use. 14.A method for treating a cancer in a subject in need thereof, comprisingadministering an effective amount of conjugated nucleic acid molecule ofclaim
 1. 15. The method according to claim 14, wherein the treatmentfurther comprises radiotherapy or chemotherapy, optionally with a DNAdamaging antitumoral agent.